WO2014050116A1 - Eustoma having cytoplasmic male sterility and method for producing said eustoma - Google Patents
Eustoma having cytoplasmic male sterility and method for producing said eustoma Download PDFInfo
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- WO2014050116A1 WO2014050116A1 PCT/JP2013/005722 JP2013005722W WO2014050116A1 WO 2014050116 A1 WO2014050116 A1 WO 2014050116A1 JP 2013005722 W JP2013005722 W JP 2013005722W WO 2014050116 A1 WO2014050116 A1 WO 2014050116A1
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/022—Genic fertility modification, e.g. apomixis
- A01H1/023—Male sterility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/02—Flowers
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/40—Gentianaceae, e.g. Exacum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
Definitions
- the present invention relates to a novel eustoma having cytoplasmic male sterility and a breeding method thereof. More specifically, the present invention relates to a novel cytoplasmic male sterility eustasis and breeding method thereof characterized by substantially lacking a pollination function due to insufficient formation of stamens or pollen.
- Eustoma is a self-propagating seed breeding plant belonging to the genus Eustoma, which grows naturally in the southern part of North America to the northern part of Central America.
- Eustoma grandiflorum English name Prairie gentian, former scientific name E. russellianum (Hook) G. Don ex Sweet, Lisianth (i) us russellianus Hook.
- Eustoma exaltatum English name Seaside gentian, Catchfly gentian, former scientific name E. selenifolium Salisb.
- It is also known as Eustoma grandiflorum or Lisianthus. In 1835, a native species was brought to Scotland and named Lisianthus russellianus Hook.
- Eustoma grandiflorum is mainly used for breeding. Eustoma is known as a flower plant with high ornamental value and market value, and in particular, varieties with various traits have been created in varieties for cut flowers, and it is counted as one of the main items of cut flowers (Non-patent literature). 1).
- hybrid first-generation crossing (F1) plants having a trait superior to their parents using hybrid strength are used. Even in the cultivated varieties of Eustoma, which are in high demand as cut flowers, hybrid first-generation hybrid varieties are the mainstream in order to provide disease resistance and high quality. Moreover, in order to increase the efficiency of sowing work in production, growers are required to supply seeds with high purity.
- the first hybrid hybrid cultivar is collected by manually pollinating pollen parent pollen to the stigma of the emasculated seed parent after manually desemination of the seed parent. Usually, the period during which the seed parent can be emasculated is limited to the period immediately before flowering and is easily affected by the weather.
- Non-patent Document 2 A fertile eustoma transformed with the rolC gene introduced by Agrobacterium rhizogenes is known (Non-patent Document 2), but the flowers are downsized, the pollen fertility disappears, and the loss of apical bud dominance ( rol syndrome) has also been reported to occur simultaneously.
- Patent Document 1 A method for producing F1 varieties by male sterile Eustoma maintained by adventitious bud regeneration by applying the technique of Non-Patent Document 2 is disclosed (Patent Document 1). However, since the F1 cultivar produced according to the method has an unintended trait, the method has a problem that it cannot be used for breeding cut flower varieties that require commercial form and height.
- Cytoplasmic male sterility which is one of male sterility, is cytoplasmically inherited. Therefore, when a cytoplasmic male sterility line and a male sterile line are crossed, the progeny plant has male sterility. High seed supply is possible. In addition, the cytoplasmic male sterile line can be easily maintained and propagated by crossing with a maintenance line having the same nuclear genome as that of the line and a normal cytoplasm. Therefore, the method for collecting hybrid first-generation hybrid varieties using cytoplasmic male sterility is extremely practical and is used in many major horticultural crops.
- Non-patent Document 3 Eustoma is known as a variety with good flower life, having a deformed pistil that prevents pollination and slows flower senescence by keeping the stigma portion closed (Patent Document 2).
- Patent Document 2 In order to improve the longevity of the flower, a strain with short stamen yarns is bred, or a plant whose structure of plant organs is changed so that it does not physically pollinate the stigma like the above-mentioned deformed pistil is used. It was necessary to do.
- the present invention is a cytoplasm in view of problems such as troublesome male removal work at the time of F1 seed production in the conventional Eustoma varieties as described above, problems of seed quality degradation, and problems of flower retention and pollen scattering. It is intended to provide a novel eustasis having male sterility and a method for producing the same.
- the present inventors have created a new Eustoma individual having cytoplasmic male sterility, and cultivated an Eustoma variety having the cytoplasmic male sterility, and the cytoplasm thereof.
- a breeding method for obtaining hybrid first-generation hybrid varieties using male sterile yeast was completed.
- the present invention relates to a novel eustoma having cytoplasmic male sterility and a method for breeding the same, and more preferably, the formation of stamen or pollen is insufficient, so that the pollination function is substantially lost.
- the present invention relates to a novel eustoma having cytoplasmic male sterility and a breeding method thereof.
- the present invention provides the following (1) to (34).
- the cytoplasm produced from the progeny of the cross wherein the Eustoma genus plant or its progeny has the Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as the seed parent and any Eustoma genus plant as the pollen parent
- the cytoplasmic male imperfection produced from the progeny of the hybrid plant in which the Eustoma genus plant or its progeny has the Eustoma genus plant having the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as the seed parent and E. grandiflorum as the pollen parent The plant of the genus Eustoma according to any one of (1) to (3), or a progeny thereof, which is a cocoon plant.
- a callus comprising a plant of the genus Eustoma or a progeny cell thereof according to any one of (1) to (4).
- the Eustoma genus plant according to any one of (1) to (4) and (8) or a progeny thereof is used as a seed parent, and an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent.
- a method for producing a hybrid primary seed which comprises collecting a hybrid primary seed from a seed parent.
- the Eustoma genus plant described in (13) or a progeny thereof is used as a seed parent, an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent, and a hybrid first-generation seed is collected from the mated seed parent.
- a method for producing a hybrid first-generation seed An Eustoma genus plant regenerated from the callus according to (18) or a progeny thereof is used as a seed parent, and an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent.
- a method for producing a hybrid first-generation seed comprising seeding a first-generation seed.
- the cytoplasmic male sterility having the useful trait By continuously backcrossing an Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 in the cytoplasm with an Eustoma genus plant having a useful trait, the cytoplasmic male sterility having the useful trait A method of producing a plant of the genus Eustoma, characterized by expressing sex.
- Eustoma genus plant having cytoplasmic male sterility or its progeny specified by the accession number FERM BP-11506 and the Eustoma genus plant having useful traits are continuously backcrossed to have the useful traits
- cytoplasmic male sterility provided by the present invention, a high-quality seed is cultivated with good harvesting, flower retention and / or appreciation. It is possible to carry out seeding.
- FIG. 1 is a photograph of the cytoplasmic male sterile of the present invention and a male flexible male
- (A) is a male flexible strain
- (B) and (C) are cytoplasms with incomplete stamen development.
- (D) is a cytoplasmic male sterile line with no stamens).
- Fig. 2 shows the cytoplasmic male sterile Eustoma line (SSE-CMS cytoplasm: Lane 1), the wild Eustoma species (Lane 2-8) possessed by Sakata Seed Co., Ltd., and the Sakata Seed Inc. The result of performing electrophoresis using total DNA of Eustoma cultivar (lanes 9-15) as a template and performing electrophoresis is shown.
- M is a molecular weight marker.
- FIG. 3 shows the base sequences of Marker 1 and Marker 2.
- the method for nurturing male sterility having male sterility according to the present invention is characterized by selecting an eustoma having cytoplasmic male sterility from a hybrid of a plant belonging to the genus Eustoma.
- the “Eustoma genus plant” or “Eustoma” according to the present invention is a plant belonging to the genus Eustoma genus, and is a horticultural crop that is familiar in Japan under the name Eustoma.
- male sterility refers to a state in which pollen cannot be produced sufficiently due to incomplete stamen development. For example, but not limited to, an eustoma with no stamens or incomplete stamen development exhibits male sterility (see FIG. 1). “Cytoplasmic male sterility” refers to a male sterility trait inherited maternally by a gene derived from a cytoplasmic organ.
- a progeny of Eustoma plant having cytoplasmic male sterility means an Eustoma plant having cytoplasmic male sterility as a female parent (seed parent), and a Eustoma plant that can be crossed with the plant is a male. It means the next generation and next generation Eustoma plants that inherit male sterility by maternal inheritance, obtained by mating as parents (pollen parents).
- “part of plant body” includes one or more cells of the plant body or cytoplasm from one or more cells, and specifically includes flowers, leaves, stems, roots. Or organs or tissues, or cells from these organs or tissues (including protoplasts prepared from cells) or cytoplasm, or a collection of said cells or cytoplasm.
- Eustoma plants identified by the accession number FERM BP-11506 also includes plants having cytoplasmic male sterility traits that are substantially equivalent to the plants. That is, as long as cytoplasmic male sterility is maintained, for example, a mutant or gene recombinant of the Eustoma plant identified by the accession number FERMFERBP-11506 is also included in the scope.
- callus of Eustoma plant identified by accession number FERM BP-11507 is substantially equivalent to the callus in which the plant regenerated from the callus has a cytoplasmic male sterility character.
- the callus is also included in the scope. That is, as long as the regenerated plant maintains cytoplasmic male sterility, for example, a mutant or gene recombinant derived from a callus of the Eustoma genus plant identified by accession number FERM BP-11507, and cytoplasm Callus derived from mutants of Eustoma plants identified by the accession number FERM BP-11506 maintaining male sterility or genetically modified plants are also included in the scope.
- the cytoplasmic male sterile Eustoma has the following characteristics. (1) When producing a hybrid first-generation hybrid cultivar, it is not necessary to remove the seed parent, so it is not time-consuming and economical seeding is possible. (2) When crossed with a male fertile line, the progeny have male sterility, so that seeds with high purity can be supplied. (3) The strain can be easily maintained and propagated by mating with a maintenance strain having the same nuclear genome as that of the strain and a normal cytoplasm. (4) Since it does not self-pollinate, it can suppress the aging of the flower by pollination and, as a result, has a long flower. (5) Since there is no pollen, petals and clothes will not be polluted by pollen scattering.
- Method for producing cytoplasmic male sterile Eustoma In the method for producing male sterile Eustoma according to the present invention, a wild species of a plant of the genus Eustoma is mated as a female parent and Eustoma grandiflorum as a male parent. Individuals showing sterility are selected and nurtured. As an individual showing male sterility, an individual having no stamen or incomplete stamen development is selected. Furthermore, in order to confirm that male sterility is a trait inherited by cytoplasm, the male sterilized strain Eustoma is backcrossed to confirm that the progeny plant exhibits male sterility.
- Flower endurance test The evaluation of the endurance of cytoplasmic male sterile Eustoma according to the present invention can be carried out as follows.
- “flower holding” means “maintenance of a flower that has blossomed”, and therefore “flower holding is good” means that the flowering period from the beginning of flowering to the end of flowering is relatively Means "long”. Whether or not the flowering period of the eustoma is relatively long is determined by cultivating a plurality of individual male fertile and cytoplasmic male sterile eustomas under the same environmental conditions for comparative evaluation.
- the flower endurance test a method for evaluating the endurance after cut flowers will be described.
- an appropriate number of normal male fertile individuals and male sterile individuals are prepared as test materials, among the easterns cultivated under the same conditions.
- Each individual is flowered with a floral pattern at the same time on the flowering day (the day when the petal opening is recognized), and the floral part is adjusted to a certain length (for example, about 4 to 6 cm).
- the prepared test material is then immersed in water with the stem cross-section immersed in water, at a constant temperature (preferably 18-22 ° C.), constant humidity (preferably 55-65%), and brightness is 12 hours. Leave in a temperature-controlled room where you can switch between and observe the appearance change of flowers.
- “Beginning of flowering” is defined as the time when the petal has opened, and “End of flowering” is determined and determined from the appearance of the petals (wrinkles and wrinkles of the petals), and the flowering period (from the beginning of flowering to the end of flowering) Period). If the average value of the measured flowering period is calculated
- cytoplasmic male sterile Eustoma strains prepared according to the present invention The sequence of mitochondrial genome is compared between cytoplasmic male sterile Eustoma strains prepared according to the present invention and the conventionally known Eustoma plants, and regions specific to cytoplasmic male sterile Eustoma strains are identified. By identifying, a molecular marker that becomes a labeling factor can be prepared. The detection of the molecular marker can be performed according to a method well known to those skilled in the art, such as a PCR method. By using molecular markers, the cytoplasmic male sterile Eustoma line according to the present invention is clearly different from conventionally known Eustoma plants not only by morphological characteristics but also by molecular biological characteristics. Can prove.
- the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 can be used as molecular markers for identifying the cytoplasmic male sterile Eustoma strain of the present invention.
- Eustoma strains having a base sequence in which one or several bases are deleted, substituted, or added in the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 are also included in SEQ ID NO: 1 or SEQ ID NO: As long as it is amplified and detected in the same manner as the base sequence of No. 2, it can be regarded as a strain substantially identical to the cytoplasmic male sterile Eustoma strain of the present invention.
- bases having a homology of 80%, preferably 90%, more preferably 95% or more with the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 An array can be used.
- Cytoplasmic male sterile Eustoma lines produced according to the present invention can be propagated asexually by tissue culture methods. For example, it can be asexually propagated by the method of inducing and redifferentiating callus disclosed in Non-Patent Document 4.
- callus induction is performed by sterilizing the surface of leaf pieces of cytoplasmic male sterile Eustoma strains grown in a greenhouse and placing them on a callus induction medium for culture. Thereafter, the formed callus is transferred to a regeneration medium and cultured to induce shoots. Next, the formed shoot is transferred to a rooting medium to induce rooting and regenerate the plant body.
- Culture conditions for callus induction, growth, and redifferentiation can be appropriately set according to known techniques by those skilled in the art.
- the male sterility of the present invention is a hybrid of a wild Eustoma genus plant whose species name was introduced from the United States and Eustoma grandiflorum. And was created at the Seed Misato Proving Ground in Sakata.
- the F2 generation seeds were sown and cultivated by about 50 to 100 individuals, and the phenotypes contained in the F2 population were confirmed. As a result, the size of flower diameter, flower color, flower type, and early and late segregation were observed from most F2 populations. Among them, the species of wild Eustoma that Sakata Seed possession was not specified. Unexpectedly, among the F2 populations derived from F1 seeds (hereinafter referred to as group A) that were crossed with E-1 of the plant as the female parent and G-1 (E. grandiflorum) of the parental line as the male parent A number of individuals exhibiting male sterility traits that were not known in the Eustoma genus have appeared.
- Progeny seeds (F2BC1 generation, each pair) selected from one individual and crossed with 4 parent lines G-2, G-3, G-4, G-5 owned by Sakata Seed Co., Ltd. as pollen parents B, C, D, E).
- the generations are written as BC1, BC2,... Based on the number of times E. grandiflorum was backcrossed to the CMS line.
- F2BC1 generation 4 individuals from set C (hereinafter referred to as ms-1), F2BC1 generation 2 individuals from set D (hereinafter referred to as ms-2), F2BC1 generation 3 individuals from set E (hereinafter ms-3) Selected) and 4 individuals (hereinafter referred to as ms-4), parent line G-3 and parent line G-6 to ms-1, parent line G-4 and parent line G-7 to ms-2,
- the parental line G-5 and the parental line G-8 were crossed with ms-3, and the parental line G-5 and the parental line G-9 were crossed with ms-4 as pollen parents to obtain progeny seeds (F2BC2 generation), respectively.
- Progeny seeds were sown and the phenotypic characteristics of the progeny were confirmed. As a result, the development of the stamen was incomplete in a part of the progeny population of the line in which G-3 and G-6 were crossed to ms-1. Of male sterile individuals, but the other combinations showed male sterile characteristics with no stamens. About 100 parent lines were crossed with these male sterile individuals to obtain progeny seeds (F2BC3 generation).
- Example 2 Confirmation of reproducibility of male sterility
- the aforementioned Eustoma genus plant E-1 was used as the female parent and the parent strain G-10 (E. grandiflorum) as the male.
- Mating as a parent F1 seeds (hereinafter referred to as “set F”) were collected.
- the above-mentioned set A and set F were sown, and 6 individuals were selected and group mating was carried out.
- the set A-derived individuals were 56 kg, 0.22 g
- the set F-derived individuals were 59 kg, with a yield of 0.47 g. Generational seeds were obtained.
- F2 generation derived from set A was 19 out of 92 individuals, and F2 generation derived from set F was male sterile in 14 out of 88 individuals The traits were shown. From the above results, it was shown that male sterility traits appear stably.
- Example 3 Elongation test of Eustoma varieties having male sterility Fermentation test of male sterility line and varieties were carried out.
- Test materials The two types of Eustoma used as test materials were F1 individuals of normal male malleable and F1 individuals of cytoplasmic male sterile Eustoma. The above two types of eustoma were collected from the eustoma field that appeared to have blossomed on the day, and the floral portion was adjusted to a length of 4 cm. The male cute Eustoma was forced to pollinate the stigma for transportation.
- cytoplasmic male sterile Eustoma was subjected to the test for individuals that were forcibly pollinated with male pollen pollen and for individuals that were not pollinated.
- test materials are inserted into a test tube filled with tap water, and a general fluorescent lamp (eyeline white rapid 40W) is turned on at a temperature of 20 ° C and a humidity of 60% ( ⁇ 5%). ⁇ The test was conducted in a temperature-controlled room that switched off every 12 hours. During the test, water was not contaminated, so water was not exchanged.
- a general fluorescent lamp eyeline white rapid 40W
- Example 4 Preparation of Markers for Discriminating SSE-CMS Eustoma Lines
- SSE-CMS Eustoma lines prepared according to the present invention exhibited phenotypic characteristics that did not exist in conventional cultivated and wild species.
- a molecular marker for identifying the SSE-CMS Eustoma line was prepared in order to prove that it is a line different from the conventionally known Eustoma genus plant.
- PCR analysis was performed using the SSE-CMS Eustoma line and the existing Eustoma line as templates, and we were able to find markers with different sizes of amplified DNA fragments. . From these polymorphic markers, nad4L / orf25 and nad7 / 4-5 were selected and analyzed for the nucleotide sequence of the amplified fragment. As a result, a region specific to the SSE-CMS Eustoma strain was identified. We were able to.
- Marker 1 is a marker that amplifies the DNA fragment of 323 bp described in SEQ ID NO: 1 using primers orf25-F and orf25-R (FIG. 3).
- Marker 2 is a marker that amplifies the 492 bp DNA fragment described in SEQ ID NO: 2 using primers nad7-F and nad7-R (FIG. 3).
- Marker2 Marker specific to SSE-CMS Eustoma line PCR was performed using the nad7-F and nad7-R primers (heat denaturation at 94 ° C for 1 minute, annealing at 65 ° C for 1 minute, extension reaction at 72 ° C for 1 minute for 30 cycles), and the nucleotide sequence shown in SEQ ID NO: 2 (492 bp ) was amplified.
- (C) nad5 / 4-5 a marker common to all Eustoma species Perform PCR (thermal denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, extension reaction at 72 ° C for 1 minute for 30 cycles) using nad5 / 4 and nad5 / 5 primers, and a DNA fragment of about 1.5 kb from all strains was amplified. That is, it has been clarified that the SSE-CMS Eustoma line produced by the present invention is a line different from conventionally known Eustoma plants.
- Example 5 SSE-CMS Eustoma Line Callus Induction, Proliferation, and Redifferentiation
- the leaf pieces were surface sterilized with 1% sodium hypochlorite solution for 10 minutes and rinsed with sterile water. Sterilized leaf pieces were placed on MS medium supplemented with 1.5 mg / l NAA to induce callus.
- Calli were transferred to B5 medium supplemented with 0.5 mg / l GA3 and 1.5 mg / l BA to induce shoots.
- the formed shoots were transferred to a B5 medium containing no plant hormone and rooted to regenerate the plant body and confirmed to be male sterile.
- the yeast and its F1 varieties that are excellent in seeding, flower retention and appreciation are bred and the high-quality seeds are seeded. make it possible to do.
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Abstract
The present invention pertains to a novel eustoma having cytoplasmic male sterility and to a method for breeding same. More specifically, the present invention pertains to a novel eustoma having cytoplasmic male sterility, characterized by being substantially devoid of pollination capacity due to insufficient stamen or pollen formation; and to a method for breeding the novel eustoma having cytoplasmic male sterility.
Description
本発明は、細胞質雄性不稔性を有する新規なユーストマ及びその育種方法に関する。さらに詳しくは、雄ずい又は、花粉の形成が不十分であるため、実質的に授粉機能を欠失することを特徴とする、新規な細胞質雄性不稔性を有するユーストマ及びその育種方法に関する。
The present invention relates to a novel eustoma having cytoplasmic male sterility and a breeding method thereof. More specifically, the present invention relates to a novel cytoplasmic male sterility eustasis and breeding method thereof characterized by substantially lacking a pollination function due to insufficient formation of stamens or pollen.
ユーストマは北アメリカ南部から中央アメリカ北部に自生するリンドウ科ユーストマ（Eustoma）属の自殖性種子繁殖植物で、（1）Eustoma grandiflorum（英名Prairie gentian, 旧学名E. russellianum（Hook）G.Don ex Sweet, Lisianth（i）us russellianus Hook.）、及び（2）Eustoma exaltatum（英名Seaside gentian, Catchfly gentian, 旧学名E. selenifolium Salisb.）の2種が存在する。別名トルコギキョウ、またはリシアンサス（Lisianthus）として親しまれている。1835年に自生種がスコットランドにもたらされ、Lisianthus russellianus Hook.と命名された。日本には、1930年代に導入され、以降主に切花用、鉢物用として品種改良が盛んに行われている。品種改良には、Eustoma grandiflorumが主に使用されている。
ユーストマは観賞価値および市場価値が高い花卉植物として知られており、特に切花用品種においては多種多様な形質を有する品種が作出されており、切花の主要品目の一つに数えられる（非特許文献1参照）。 Eustoma is a self-propagating seed breeding plant belonging to the genus Eustoma, which grows naturally in the southern part of North America to the northern part of Central America. (1) Eustoma grandiflorum (English name Prairie gentian, former scientific name E. russellianum (Hook) G. Don ex Sweet, Lisianth (i) us russellianus Hook.) And (2) Eustoma exaltatum (English name Seaside gentian, Catchfly gentian, former scientific name E. selenifolium Salisb.). It is also known as Eustoma grandiflorum or Lisianthus. In 1835, a native species was brought to Scotland and named Lisianthus russellianus Hook. In Japan, it was introduced in the 1930s, and since then, varieties have been actively improved mainly for cut flowers and pots. Eustoma grandiflorum is mainly used for breeding.
Eustoma is known as a flower plant with high ornamental value and market value, and in particular, varieties with various traits have been created in varieties for cut flowers, and it is counted as one of the main items of cut flowers (Non-patent literature). 1).
ユーストマは観賞価値および市場価値が高い花卉植物として知られており、特に切花用品種においては多種多様な形質を有する品種が作出されており、切花の主要品目の一つに数えられる（非特許文献1参照）。 Eustoma is a self-propagating seed breeding plant belonging to the genus Eustoma, which grows naturally in the southern part of North America to the northern part of Central America. (1) Eustoma grandiflorum (English name Prairie gentian, former scientific name E. russellianum (Hook) G. Don ex Sweet, Lisianth (i) us russellianus Hook.) And (2) Eustoma exaltatum (English name Seaside gentian, Catchfly gentian, former scientific name E. selenifolium Salisb.). It is also known as Eustoma grandiflorum or Lisianthus. In 1835, a native species was brought to Scotland and named Lisianthus russellianus Hook. In Japan, it was introduced in the 1930s, and since then, varieties have been actively improved mainly for cut flowers and pots. Eustoma grandiflorum is mainly used for breeding.
Eustoma is known as a flower plant with high ornamental value and market value, and in particular, varieties with various traits have been created in varieties for cut flowers, and it is counted as one of the main items of cut flowers (Non-patent literature). 1).
一般的に、主要園芸作物においては、雑種強勢を利用した、両親よりも優れた形質を有する雑種第一代交配（F1）植物の利用が行われている。
切花としての需要が高いユーストマの栽培品種においても、耐病性や高品質性を持たせるため、雑種第一代交配品種が主流である。
また、生産における播種作業を効率化するため、育成者には純度の高い種子の供給が求められている。
雑種第一代交配品種の採種は、まず人手による種子親の除雄後、花粉親の花粉を、除雄した種子親の柱頭へ授粉させることにより行われる。
通常、種子親を除雄できる期間は開花直前期に限定され、かつ天候に左右されやすいため、除雄には、除雄可能な期間の予測がつき難いという作業性の問題がある。また、この作業は人手により行われるため、除雄しきれずに残存した花粉が原因となって意図せぬ自家受粉種子が発生する場合があり、自家受粉種子の混入による種子の品質の低下が問題となっていた。
上記の理由から、ユーストマでは、人手による種子親の除雄が不要である、雄性不稔性を利用した採種方法の開発が望まれている。 In general, in primary horticultural crops, hybrid first-generation crossing (F1) plants having a trait superior to their parents using hybrid strength are used.
Even in the cultivated varieties of Eustoma, which are in high demand as cut flowers, hybrid first-generation hybrid varieties are the mainstream in order to provide disease resistance and high quality.
Moreover, in order to increase the efficiency of sowing work in production, growers are required to supply seeds with high purity.
The first hybrid hybrid cultivar is collected by manually pollinating pollen parent pollen to the stigma of the emasculated seed parent after manually desemination of the seed parent.
Usually, the period during which the seed parent can be emasculated is limited to the period immediately before flowering and is easily affected by the weather. Therefore, there is a problem in workability that it is difficult to predict the period during which masculinization is possible. In addition, since this work is performed manually, undesired self-pollinated seeds may be generated due to the remaining pollen that cannot be removed, and deterioration of the seed quality due to mixing of self-pollinated seeds is a problem. It was.
For the above reasons, it has been desired for the Eustoma to develop a seeding method using male sterility, which does not require manual seed parenting.
切花としての需要が高いユーストマの栽培品種においても、耐病性や高品質性を持たせるため、雑種第一代交配品種が主流である。
また、生産における播種作業を効率化するため、育成者には純度の高い種子の供給が求められている。
雑種第一代交配品種の採種は、まず人手による種子親の除雄後、花粉親の花粉を、除雄した種子親の柱頭へ授粉させることにより行われる。
通常、種子親を除雄できる期間は開花直前期に限定され、かつ天候に左右されやすいため、除雄には、除雄可能な期間の予測がつき難いという作業性の問題がある。また、この作業は人手により行われるため、除雄しきれずに残存した花粉が原因となって意図せぬ自家受粉種子が発生する場合があり、自家受粉種子の混入による種子の品質の低下が問題となっていた。
上記の理由から、ユーストマでは、人手による種子親の除雄が不要である、雄性不稔性を利用した採種方法の開発が望まれている。 In general, in primary horticultural crops, hybrid first-generation crossing (F1) plants having a trait superior to their parents using hybrid strength are used.
Even in the cultivated varieties of Eustoma, which are in high demand as cut flowers, hybrid first-generation hybrid varieties are the mainstream in order to provide disease resistance and high quality.
Moreover, in order to increase the efficiency of sowing work in production, growers are required to supply seeds with high purity.
The first hybrid hybrid cultivar is collected by manually pollinating pollen parent pollen to the stigma of the emasculated seed parent after manually desemination of the seed parent.
Usually, the period during which the seed parent can be emasculated is limited to the period immediately before flowering and is easily affected by the weather. Therefore, there is a problem in workability that it is difficult to predict the period during which masculinization is possible. In addition, since this work is performed manually, undesired self-pollinated seeds may be generated due to the remaining pollen that cannot be removed, and deterioration of the seed quality due to mixing of self-pollinated seeds is a problem. It was.
For the above reasons, it has been desired for the Eustoma to develop a seeding method using male sterility, which does not require manual seed parenting.
アグロバクテリウム・リゾゲネス（Agrobacterium rhizogenes）によりrolC遺伝子を導入し、形質転換した矮性トルコギキョウが知られているが（非特許文献2）、花が小型化し、花粉稔性がなくなり頂芽優勢の消失（rolシンドローム）も同時に起こることが報告されている。
非特許文献2の技術を応用し、不定芽再生により維持されている雄性不稔のトルコギキョウによるF1品種を作出する方法が開示されている（特許文献1）。しかし当該方法に従い作出されたF1品種は目的としない形質を伴うため、当該方法には商品性がある草姿や草丈を要求される切花品種の育種に利用することができないという問題がある。 A fertile eustoma transformed with the rolC gene introduced by Agrobacterium rhizogenes is known (Non-patent Document 2), but the flowers are downsized, the pollen fertility disappears, and the loss of apical bud dominance ( rol syndrome) has also been reported to occur simultaneously.
A method for producing F1 varieties by male sterile Eustoma maintained by adventitious bud regeneration by applying the technique of Non-PatentDocument 2 is disclosed (Patent Document 1). However, since the F1 cultivar produced according to the method has an unintended trait, the method has a problem that it cannot be used for breeding cut flower varieties that require commercial form and height.
非特許文献2の技術を応用し、不定芽再生により維持されている雄性不稔のトルコギキョウによるF1品種を作出する方法が開示されている（特許文献1）。しかし当該方法に従い作出されたF1品種は目的としない形質を伴うため、当該方法には商品性がある草姿や草丈を要求される切花品種の育種に利用することができないという問題がある。 A fertile eustoma transformed with the rolC gene introduced by Agrobacterium rhizogenes is known (Non-patent Document 2), but the flowers are downsized, the pollen fertility disappears, and the loss of apical bud dominance ( rol syndrome) has also been reported to occur simultaneously.
A method for producing F1 varieties by male sterile Eustoma maintained by adventitious bud regeneration by applying the technique of Non-Patent
雄性不稔性の１つである細胞質雄性不稔性は、細胞質遺伝するため、細胞質雄性不稔系統と雄性可稔系統とを交配した場合、後代植物は雄性不稔性を有するため、純度の高い種子の供給が可能となる。また、細胞質雄性不稔系統は、当該系統と核ゲノムが同一であって細胞質が正常である維持系統との交配により、系統の維持および増殖が容易である。従って、細胞質雄性不稔性を利用した雑種第一代交配品種の採種方法は、実用性が極めて高く、多くの主要園芸作物において利用されている。しかしながら、ユーストマにおいては、雄性不稔性を利用した採種方法の開発が望まれていたにもかかわらず、細胞質雄性不稔ユーストマの作出方法は報告されていない。よって、ユーストマF1品種の種子生産において、実用的な雄性不稔系統を用いた採種体系が存在しないため、除雄作業を要する採種が行われている。
Cytoplasmic male sterility, which is one of male sterility, is cytoplasmically inherited. Therefore, when a cytoplasmic male sterility line and a male sterile line are crossed, the progeny plant has male sterility. High seed supply is possible. In addition, the cytoplasmic male sterile line can be easily maintained and propagated by crossing with a maintenance line having the same nuclear genome as that of the line and a normal cytoplasm. Therefore, the method for collecting hybrid first-generation hybrid varieties using cytoplasmic male sterility is extremely practical and is used in many major horticultural crops. However, in Euston, there has been no report on a method for producing a cytoplasmic male sterilized yeast, although development of a seeding method utilizing male sterility has been desired. Therefore, in the seed production of Eustoma F1 varieties, there is no practical seeding system using male sterile lines, and so seeding that requires male removal work is performed.
一方、花弁の老化を遅らせることによって、花の観賞期間を延長する花持ちの良さが求められている。花弁の老化の原因として、受粉、または雌ずいの柱頭や花柱が傷害を受けることによるエチレン生成などが挙げられる（非特許文献3）。
ユーストマでは、花持ちの良い品種として、柱頭の部分が閉じたままであることにより、受粉することを妨げ花の老化を遅くする変形雌ずいを有するユーストマが知られている（特許文献2）。
花持ちを良くするためには、雄ずいの花糸が短い系統を育成するか、又は前述の変形雌ずいのように物理的に柱頭に受粉しないよう植物の器官の構造を変えた系統を利用することが必要であった。 On the other hand, there is a demand for good flower lasting that extends the period of flower viewing by delaying senescence of petals. The causes of petal aging include pollination or ethylene production due to damage to pistil stigmas and styles (Non-patent Document 3).
Eustoma is known as a variety with good flower life, having a deformed pistil that prevents pollination and slows flower senescence by keeping the stigma portion closed (Patent Document 2).
In order to improve the longevity of the flower, a strain with short stamen yarns is bred, or a plant whose structure of plant organs is changed so that it does not physically pollinate the stigma like the above-mentioned deformed pistil is used. It was necessary to do.
ユーストマでは、花持ちの良い品種として、柱頭の部分が閉じたままであることにより、受粉することを妨げ花の老化を遅くする変形雌ずいを有するユーストマが知られている（特許文献2）。
花持ちを良くするためには、雄ずいの花糸が短い系統を育成するか、又は前述の変形雌ずいのように物理的に柱頭に受粉しないよう植物の器官の構造を変えた系統を利用することが必要であった。 On the other hand, there is a demand for good flower lasting that extends the period of flower viewing by delaying senescence of petals. The causes of petal aging include pollination or ethylene production due to damage to pistil stigmas and styles (Non-patent Document 3).
Eustoma is known as a variety with good flower life, having a deformed pistil that prevents pollination and slows flower senescence by keeping the stigma portion closed (Patent Document 2).
In order to improve the longevity of the flower, a strain with short stamen yarns is bred, or a plant whose structure of plant organs is changed so that it does not physically pollinate the stigma like the above-mentioned deformed pistil is used. It was necessary to do.
さらに、市場価値の高い花卉植物の条件の１つとして、花粉の飛散を防止することが求められている。しかし、現在のユーストマ品種では、いずれの品種においても雄ずいは花粉を有するため、花粉の飛散による花弁または衣服の汚損などの課題があった。
Furthermore, as one of the conditions of flower plants with high market value, it is required to prevent pollen from scattering. However, in the current Eustoma varieties, the stamens have pollen in all varieties, and thus there have been problems such as petal or clothing contamination due to pollen scattering.
雄性不稔性を利用することにより、有用形質に変化を及ぼすことなく、花持ち性等の課題を解決することが可能となるが、そのような実用的な雄性不稔性を有するユーストマは作出されていない。
By utilizing male sterility, it is possible to solve problems such as flower retention without affecting useful traits, but we have created a practical sterility with male sterility. It has not been.
本発明は、上述のような従来のユーストマ品種におけるF1種子生産時の煩雑な除雄作業等の問題点、種子品質低下の問題点、更には花持ち性や花粉飛散の課題に鑑みて、細胞質雄性不稔性を有する新規なユーストマ及びその作出方法を提供するものである。
The present invention is a cytoplasm in view of problems such as troublesome male removal work at the time of F1 seed production in the conventional Eustoma varieties as described above, problems of seed quality degradation, and problems of flower retention and pollen scattering. It is intended to provide a novel eustasis having male sterility and a method for producing the same.
本発明者らは、上記課題を解決すべく鋭意検討を行った結果、細胞質雄性不稔性を有する新規なユーストマ個体を作出し、その細胞質雄性不稔性を有するユーストマ品種を育成し、その細胞質雄性不稔性ユーストマを用いて雑種第一代交配品種を得る育成方法を完成した。
As a result of intensive studies to solve the above-mentioned problems, the present inventors have created a new Eustoma individual having cytoplasmic male sterility, and cultivated an Eustoma variety having the cytoplasmic male sterility, and the cytoplasm thereof. A breeding method for obtaining hybrid first-generation hybrid varieties using male sterile yeast was completed.
即ち、本発明は、細胞質雄性不稔性を有する新規なユーストマ及びその育種方法に関し、さらに好ましくは、雄ずい又は、花粉の形成が不十分であるため、実質的に授粉機能を欠失することを特徴とする、新規な細胞質雄性不稔性を有するユーストマ及びその育種方法に関する。
That is, the present invention relates to a novel eustoma having cytoplasmic male sterility and a method for breeding the same, and more preferably, the formation of stamen or pollen is insufficient, so that the pollination function is substantially lost. The present invention relates to a novel eustoma having cytoplasmic male sterility and a breeding method thereof.
すなわち、本発明は、以下の（１）～（３４）を提供する。
（１）細胞質雄性不稔性を有するEustoma属植物、またはその後代。
（２）配列番号１もしくは配列番号２に記載した塩基配列を有することを特徴とする、（１）に記載のEustoma属植物、またはその後代。
（３）前記Eustoma属植物またはその後代が、配列番号１もしくは配列番号２に記載した塩基配列を有するEustoma属植物を種子親、任意のEustoma属植物を花粉親とした交雑後代から作出された細胞質雄性不稔植物であることを特徴とする、（１）または（２）に記載のEustoma属植物、またはその後代。
（４）前記Eustoma属植物またはその後代が、配列番号１もしくは配列番号２に記載した塩基配列を有するEustoma属植物を種子親、E. grandiflorumを花粉親とした交雑後代から作出された細胞質雄性不稔植物であることを特徴とする、（１）～（３）のいずれかに記載のEustoma属植物、またはその後代。
（５）（１）～（４）のいずれかに記載のEustoma属植物またはその後代の植物体の一部。
（６）（１）～（４）のいずれかに記載のEustoma属植物またはその後代の種子。
（７）（１）～（４）のいずれかに記載のEustoma属植物またはその後代の細胞を含むカルス。
（８）（７）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物、またはその後代。
（９）（８）に記載のEustoma属植物またはその後代の植物体の一部。
（１０）（７）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物を作製する方法。
（１１）（１）～（４）および（８）のいずれかに記載のEustoma属植物もしくはその後代、（５）および（９）に記載の植物体の一部、（６）に記載の種子、または（７）に記載のカルスに含まれる細胞質。
（１２）（１）～（４）および（８）のいずれかに記載のEustoma属植物もしくはその後代、（５）および（９）に記載の植物体の一部、（６）に記載の種子、または（７）に記載のカルスに含まれるミトコンドリア。
（１３）受託番号FERM BP-11506で特定される、細胞質雄性不稔性を有するEustoma属植物、またはその後代。
（１４）（１３）に記載のEustoma属植物またはその後代の植物体の一部。
（１５）（１３）に記載のEustoma属植物またはその後代の種子。
（１６）（１３）に記載のEustoma属植物もしくはその後代、（１４）に記載の植物体の一部、または（１５）に記載の種子に含まれる細胞質。
（１７）（１３）に記載のEustoma属植物もしくはその後代、（１４）に記載の植物体の一部、または（１５）に記載の種子に含まれるミトコンドリア。
（１８）受託番号FERM BP-11507で特定される、細胞質雄性不稔性を有するEustoma属植物のカルス。
（１９）（１８）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物、またはその後代。
（２０）（１９）に記載のEustoma属植物またはその後代の植物体の一部。
（２１）（１８）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物を作製する方法。
（２２）（１８）に記載のカルス、（１９）に記載のEustoma属植物もしくはその後代、または（２０）に記載の植物体の一部に含まれる細胞質。
（２３）（１８）に記載のカルス、（１９）に記載のEustoma属植物もしくはその後代、または（２０）に記載の植物体の一部に含まれるミトコンドリア。
（２４）（１）～（４）および（８）のいずれかに記載のEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。
（２５）（１３）に記載のEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。
（２６）（１８）に記載のカルスより再生させたEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。
（２７）（２４）～（２６）のいずれかに記載の方法により作出された雑種第一代種子。
（２８）（２７）に記載の雑種第一代種子から生育させた雑種第一代植物。
（２９）配列番号１もしくは配列番号２に記載した塩基配列を細胞質に有するEustoma属植物と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。
（３０）受託番号FERM BP-11506で特定される、細胞質雄性不稔性を有するEustoma属植物またはその後代と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。
（３１）受託番号FERM BP-11507で特定される、細胞質雄性不稔性を有するEustoma属植物のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物またはその後代と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。
（３２）有用形質を有するEustoma属植物がE. grandiflorumに由来するものである、（２９）～（３１）のいずれかに記載の作出方法。
（３３）（２９）～（３２）のいずれかに記載の方法により作出されたEustoma属植物、またはその後代。
（３４）（３３）に記載のEustoma属植物またはその後代の植物体の一部。 That is, the present invention provides the following (1) to (34).
(1) A plant of the genus Eustoma having cytoplasmic male sterility, or a progeny thereof.
(2) The plant of the genus Eustoma according to (1) or a progeny thereof, which has the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.
(3) The cytoplasm produced from the progeny of the cross, wherein the Eustoma genus plant or its progeny has the Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as the seed parent and any Eustoma genus plant as the pollen parent The plant of the genus Eustoma according to (1) or (2), or a progeny thereof, which is a male sterile plant.
(4) The cytoplasmic male imperfection produced from the progeny of the hybrid plant in which the Eustoma genus plant or its progeny has the Eustoma genus plant having the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as the seed parent and E. grandiflorum as the pollen parent The plant of the genus Eustoma according to any one of (1) to (3), or a progeny thereof, which is a cocoon plant.
(5) A part of the plant belonging to the genus Eustoma or a progeny plant according to any one of (1) to (4).
(6) A plant of the genus Eustoma or a seed of its progeny according to any one of (1) to (4).
(7) A callus comprising a plant of the genus Eustoma or a progeny cell thereof according to any one of (1) to (4).
(8) An Eustoma genus plant derived from the callus according to (7) and propagated asexually by tissue culture, or a progeny thereof.
(9) A part of the plant of the genus Eustoma or its progeny as described in (8).
(10) A method for producing an Eustoma genus plant derived from the callus according to (7) and propagated asexually by tissue culture.
(11) A plant of the genus Eustoma or a progeny thereof according to any one of (1) to (4) and (8), a part of the plant body according to (5) and (9), and a seed according to (6) Or the cytoplasm contained in the callus according to (7).
(12) A plant of the genus Eustoma or a progeny thereof according to any one of (1) to (4) and (8), a part of the plant body according to (5) and (9), and a seed according to (6) Or mitochondria contained in the callus according to (7).
(13) A plant belonging to the genus Eustoma having cytoplasmic male sterility identified by the accession number FERM BP-11506, or a progeny thereof.
(14) A part of the plant of the genus Eustoma or its progeny as described in (13).
(15) The Eustoma genus plant or the progeny seed thereof according to (13).
(16) The cytoplasm contained in the plant belonging to the genus Eustoma or its progeny according to (13), a part of the plant according to (14), or the seed according to (15).
(17) The Eustoma plant or its progeny according to (13), a part of the plant according to (14), or the mitochondria contained in the seed according to (15).
(18) A callus of a plant belonging to the genus Eustoma having cytoplasmic male sterility, identified by the accession number FERM BP-11507.
(19) An Eustoma genus plant derived from the callus according to (18) and propagated asexually by tissue culture, or a progeny thereof.
(20) A part of the plant of the genus Eustoma or its progeny as described in (19).
(21) A method for producing an Eustoma genus plant derived from the callus according to (18) and propagated asexually by tissue culture.
(22) The cytoplasm contained in the callus according to (18), the plant of the genus Eustoma or its progeny according to (19), or a part of the plant according to (20).
(23) The callus according to (18), the Eustoma genus plant or its progeny according to (19), or the mitochondria contained in a part of the plant according to (20).
(24) The Eustoma genus plant according to any one of (1) to (4) and (8) or a progeny thereof is used as a seed parent, and an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent. A method for producing a hybrid primary seed, which comprises collecting a hybrid primary seed from a seed parent.
(25) The Eustoma genus plant described in (13) or a progeny thereof is used as a seed parent, an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent, and a hybrid first-generation seed is collected from the mated seed parent. A method for producing a hybrid first-generation seed.
(26) An Eustoma genus plant regenerated from the callus according to (18) or a progeny thereof is used as a seed parent, and an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent. A method for producing a hybrid first-generation seed, comprising seeding a first-generation seed.
(27) A hybrid first-generation seed produced by the method according to any one of (24) to (26).
(28) A hybrid primary plant grown from the hybrid primary seed according to (27).
(29) By continuously backcrossing an Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 in the cytoplasm with an Eustoma genus plant having a useful trait, the cytoplasmic male sterility having the useful trait A method of producing a plant of the genus Eustoma, characterized by expressing sex.
(30) Eustoma genus plant having cytoplasmic male sterility or its progeny specified by the accession number FERM BP-11506 and the Eustoma genus plant having useful traits are continuously backcrossed to have the useful traits A method for producing a plant of the genus Eustoma, characterized by expressing cytoplasmic male sterility.
(31) Eustoma genus plant derived from callus of Eustoma genus plant having cytoplasmic male sterility and identified as accession number FERM BP-11507, and having a useful trait A method for producing a plant of the genus Eustoma having the above-mentioned useful character and expressing cytoplasmic male sterility by continuously backcrossing the plant of the genus Eustoma.
(32) The production method according to any of (29) to (31), wherein the plant belonging to the genus Eustoma having a useful trait is derived from E. grandiflorum.
(33) A plant of the genus Eustoma produced by the method according to any one of (29) to (32), or a progeny thereof.
(34) A part of the plant of the genus Eustoma or its progeny as described in (33).
（１）細胞質雄性不稔性を有するEustoma属植物、またはその後代。
（２）配列番号１もしくは配列番号２に記載した塩基配列を有することを特徴とする、（１）に記載のEustoma属植物、またはその後代。
（３）前記Eustoma属植物またはその後代が、配列番号１もしくは配列番号２に記載した塩基配列を有するEustoma属植物を種子親、任意のEustoma属植物を花粉親とした交雑後代から作出された細胞質雄性不稔植物であることを特徴とする、（１）または（２）に記載のEustoma属植物、またはその後代。
（４）前記Eustoma属植物またはその後代が、配列番号１もしくは配列番号２に記載した塩基配列を有するEustoma属植物を種子親、E. grandiflorumを花粉親とした交雑後代から作出された細胞質雄性不稔植物であることを特徴とする、（１）～（３）のいずれかに記載のEustoma属植物、またはその後代。
（５）（１）～（４）のいずれかに記載のEustoma属植物またはその後代の植物体の一部。
（６）（１）～（４）のいずれかに記載のEustoma属植物またはその後代の種子。
（７）（１）～（４）のいずれかに記載のEustoma属植物またはその後代の細胞を含むカルス。
（８）（７）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物、またはその後代。
（９）（８）に記載のEustoma属植物またはその後代の植物体の一部。
（１０）（７）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物を作製する方法。
（１１）（１）～（４）および（８）のいずれかに記載のEustoma属植物もしくはその後代、（５）および（９）に記載の植物体の一部、（６）に記載の種子、または（７）に記載のカルスに含まれる細胞質。
（１２）（１）～（４）および（８）のいずれかに記載のEustoma属植物もしくはその後代、（５）および（９）に記載の植物体の一部、（６）に記載の種子、または（７）に記載のカルスに含まれるミトコンドリア。
（１３）受託番号FERM BP-11506で特定される、細胞質雄性不稔性を有するEustoma属植物、またはその後代。
（１４）（１３）に記載のEustoma属植物またはその後代の植物体の一部。
（１５）（１３）に記載のEustoma属植物またはその後代の種子。
（１６）（１３）に記載のEustoma属植物もしくはその後代、（１４）に記載の植物体の一部、または（１５）に記載の種子に含まれる細胞質。
（１７）（１３）に記載のEustoma属植物もしくはその後代、（１４）に記載の植物体の一部、または（１５）に記載の種子に含まれるミトコンドリア。
（１８）受託番号FERM BP-11507で特定される、細胞質雄性不稔性を有するEustoma属植物のカルス。
（１９）（１８）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物、またはその後代。
（２０）（１９）に記載のEustoma属植物またはその後代の植物体の一部。
（２１）（１８）に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物を作製する方法。
（２２）（１８）に記載のカルス、（１９）に記載のEustoma属植物もしくはその後代、または（２０）に記載の植物体の一部に含まれる細胞質。
（２３）（１８）に記載のカルス、（１９）に記載のEustoma属植物もしくはその後代、または（２０）に記載の植物体の一部に含まれるミトコンドリア。
（２４）（１）～（４）および（８）のいずれかに記載のEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。
（２５）（１３）に記載のEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。
（２６）（１８）に記載のカルスより再生させたEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。
（２７）（２４）～（２６）のいずれかに記載の方法により作出された雑種第一代種子。
（２８）（２７）に記載の雑種第一代種子から生育させた雑種第一代植物。
（２９）配列番号１もしくは配列番号２に記載した塩基配列を細胞質に有するEustoma属植物と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。
（３０）受託番号FERM BP-11506で特定される、細胞質雄性不稔性を有するEustoma属植物またはその後代と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。
（３１）受託番号FERM BP-11507で特定される、細胞質雄性不稔性を有するEustoma属植物のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物またはその後代と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。
（３２）有用形質を有するEustoma属植物がE. grandiflorumに由来するものである、（２９）～（３１）のいずれかに記載の作出方法。
（３３）（２９）～（３２）のいずれかに記載の方法により作出されたEustoma属植物、またはその後代。
（３４）（３３）に記載のEustoma属植物またはその後代の植物体の一部。 That is, the present invention provides the following (1) to (34).
(1) A plant of the genus Eustoma having cytoplasmic male sterility, or a progeny thereof.
(2) The plant of the genus Eustoma according to (1) or a progeny thereof, which has the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.
(3) The cytoplasm produced from the progeny of the cross, wherein the Eustoma genus plant or its progeny has the Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as the seed parent and any Eustoma genus plant as the pollen parent The plant of the genus Eustoma according to (1) or (2), or a progeny thereof, which is a male sterile plant.
(4) The cytoplasmic male imperfection produced from the progeny of the hybrid plant in which the Eustoma genus plant or its progeny has the Eustoma genus plant having the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as the seed parent and E. grandiflorum as the pollen parent The plant of the genus Eustoma according to any one of (1) to (3), or a progeny thereof, which is a cocoon plant.
(5) A part of the plant belonging to the genus Eustoma or a progeny plant according to any one of (1) to (4).
(6) A plant of the genus Eustoma or a seed of its progeny according to any one of (1) to (4).
(7) A callus comprising a plant of the genus Eustoma or a progeny cell thereof according to any one of (1) to (4).
(8) An Eustoma genus plant derived from the callus according to (7) and propagated asexually by tissue culture, or a progeny thereof.
(9) A part of the plant of the genus Eustoma or its progeny as described in (8).
(10) A method for producing an Eustoma genus plant derived from the callus according to (7) and propagated asexually by tissue culture.
(11) A plant of the genus Eustoma or a progeny thereof according to any one of (1) to (4) and (8), a part of the plant body according to (5) and (9), and a seed according to (6) Or the cytoplasm contained in the callus according to (7).
(12) A plant of the genus Eustoma or a progeny thereof according to any one of (1) to (4) and (8), a part of the plant body according to (5) and (9), and a seed according to (6) Or mitochondria contained in the callus according to (7).
(13) A plant belonging to the genus Eustoma having cytoplasmic male sterility identified by the accession number FERM BP-11506, or a progeny thereof.
(14) A part of the plant of the genus Eustoma or its progeny as described in (13).
(15) The Eustoma genus plant or the progeny seed thereof according to (13).
(16) The cytoplasm contained in the plant belonging to the genus Eustoma or its progeny according to (13), a part of the plant according to (14), or the seed according to (15).
(17) The Eustoma plant or its progeny according to (13), a part of the plant according to (14), or the mitochondria contained in the seed according to (15).
(18) A callus of a plant belonging to the genus Eustoma having cytoplasmic male sterility, identified by the accession number FERM BP-11507.
(19) An Eustoma genus plant derived from the callus according to (18) and propagated asexually by tissue culture, or a progeny thereof.
(20) A part of the plant of the genus Eustoma or its progeny as described in (19).
(21) A method for producing an Eustoma genus plant derived from the callus according to (18) and propagated asexually by tissue culture.
(22) The cytoplasm contained in the callus according to (18), the plant of the genus Eustoma or its progeny according to (19), or a part of the plant according to (20).
(23) The callus according to (18), the Eustoma genus plant or its progeny according to (19), or the mitochondria contained in a part of the plant according to (20).
(24) The Eustoma genus plant according to any one of (1) to (4) and (8) or a progeny thereof is used as a seed parent, and an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent. A method for producing a hybrid primary seed, which comprises collecting a hybrid primary seed from a seed parent.
(25) The Eustoma genus plant described in (13) or a progeny thereof is used as a seed parent, an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent, and a hybrid first-generation seed is collected from the mated seed parent. A method for producing a hybrid first-generation seed.
(26) An Eustoma genus plant regenerated from the callus according to (18) or a progeny thereof is used as a seed parent, and an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent. A method for producing a hybrid first-generation seed, comprising seeding a first-generation seed.
(27) A hybrid first-generation seed produced by the method according to any one of (24) to (26).
(28) A hybrid primary plant grown from the hybrid primary seed according to (27).
(29) By continuously backcrossing an Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 in the cytoplasm with an Eustoma genus plant having a useful trait, the cytoplasmic male sterility having the useful trait A method of producing a plant of the genus Eustoma, characterized by expressing sex.
(30) Eustoma genus plant having cytoplasmic male sterility or its progeny specified by the accession number FERM BP-11506 and the Eustoma genus plant having useful traits are continuously backcrossed to have the useful traits A method for producing a plant of the genus Eustoma, characterized by expressing cytoplasmic male sterility.
(31) Eustoma genus plant derived from callus of Eustoma genus plant having cytoplasmic male sterility and identified as accession number FERM BP-11507, and having a useful trait A method for producing a plant of the genus Eustoma having the above-mentioned useful character and expressing cytoplasmic male sterility by continuously backcrossing the plant of the genus Eustoma.
(32) The production method according to any of (29) to (31), wherein the plant belonging to the genus Eustoma having a useful trait is derived from E. grandiflorum.
(33) A plant of the genus Eustoma produced by the method according to any one of (29) to (32), or a progeny thereof.
(34) A part of the plant of the genus Eustoma or its progeny as described in (33).
本発明により提供される新規な細胞質雄性不稔性を有するユーストマを利用することにより、採種性、花持ち性、および／または鑑賞性に優れたユーストマとそのF１品種を育成し、その高品質種子の採種を行うことを可能とする。
By using the novel cytoplasmic male sterility provided by the present invention, a high-quality seed is cultivated with good harvesting, flower retention and / or appreciation. It is possible to carry out seeding.
以下、本発明について詳細に説明する。
１．細胞質雄性不稔ユーストマ
本発明の雄性不稔性を有するユーストマの育成方法は、ユーストマ属植物の雑種から細胞質雄性不稔性を有するユーストマを選抜することを特徴とする。
本発明に係る「ユーストマ属植物」または「ユーストマ」とは、リンドウ科ユーストマ（Eustoma）属の植物であって、日本ではトルコギキョウの名前で親しまれている園芸作物である。 Hereinafter, the present invention will be described in detail.
1. Cytoplasmic Male Sterile Eustoma The method for nurturing male sterility having male sterility according to the present invention is characterized by selecting an eustoma having cytoplasmic male sterility from a hybrid of a plant belonging to the genus Eustoma.
The “Eustoma genus plant” or “Eustoma” according to the present invention is a plant belonging to the genus Eustoma genus, and is a horticultural crop that is familiar in Japan under the name Eustoma.
１．細胞質雄性不稔ユーストマ
本発明の雄性不稔性を有するユーストマの育成方法は、ユーストマ属植物の雑種から細胞質雄性不稔性を有するユーストマを選抜することを特徴とする。
本発明に係る「ユーストマ属植物」または「ユーストマ」とは、リンドウ科ユーストマ（Eustoma）属の植物であって、日本ではトルコギキョウの名前で親しまれている園芸作物である。 Hereinafter, the present invention will be described in detail.
1. Cytoplasmic Male Sterile Eustoma The method for nurturing male sterility having male sterility according to the present invention is characterized by selecting an eustoma having cytoplasmic male sterility from a hybrid of a plant belonging to the genus Eustoma.
The “Eustoma genus plant” or “Eustoma” according to the present invention is a plant belonging to the genus Eustoma genus, and is a horticultural crop that is familiar in Japan under the name Eustoma.
ユーストマをはじめ、通常花にはがく片と花弁（花びら）があり、その内側に雄ずい（雄しべ）と雌ずい（雌しべ）がある。典型的な雄ずいは、花粉を含む葯と花糸とからなる。本発明にかかる、「雄性不稔」とは、雄ずいの発達が不完全なため、花粉を充分に産出できない状態を示す。限定するものではないが、例えば、雄ずいが全く存在しない状態や、雄ずいの発達が不完全な状態のユーストマは、雄性不稔性を示す（図1参照）。また、「細胞質雄性不稔」とは、細胞質の器官由来の遺伝子によって母性遺伝する雄性不稔形質のことをいう。
◎ There are sepals and petals (petals) on the flowers, including the eustoma, and there are stamens (stamens) and pistils (pistils) inside. A typical stamen consists of pods and pollen containing pollen. The term “male sterility” according to the present invention refers to a state in which pollen cannot be produced sufficiently due to incomplete stamen development. For example, but not limited to, an eustoma with no stamens or incomplete stamen development exhibits male sterility (see FIG. 1). “Cytoplasmic male sterility” refers to a male sterility trait inherited maternally by a gene derived from a cytoplasmic organ.
本発明において「細胞質雄性不稔性を有するユーストマ属植物の後代」とは、細胞質雄性不稔性を有するユーストマ属植物を雌親（種子親）とし、該植物に交配可能なユーストマ属植物を雄親（花粉親）として交配することによって得られる、母性遺伝により雄性不稔性を引き継ぐ次世代及び次世代以降のユーストマ属植物を意味する。また、本発明において「植物体の一部」とは、当該植物体の１個以上の細胞または１個以上の細胞からの細胞質を含むものであり、具体的には花、葉、茎、根等の器官または組織、或いは、これらの器官または組織からの細胞（細胞から調製されたプロトプラストを含む）もしくは細胞質、或いは前記細胞もしくは細胞質の集合体を意味する。
In the present invention, “a progeny of Eustoma plant having cytoplasmic male sterility” means an Eustoma plant having cytoplasmic male sterility as a female parent (seed parent), and a Eustoma plant that can be crossed with the plant is a male. It means the next generation and next generation Eustoma plants that inherit male sterility by maternal inheritance, obtained by mating as parents (pollen parents). In the present invention, “part of plant body” includes one or more cells of the plant body or cytoplasm from one or more cells, and specifically includes flowers, leaves, stems, roots. Or organs or tissues, or cells from these organs or tissues (including protoplasts prepared from cells) or cytoplasm, or a collection of said cells or cytoplasm.
本明細書において、「受託番号FERM BP-11506で特定されるユーストマ属植物」には、細胞質雄性不稔性の形質を有する、該植物と実質的に等価な植物もその範囲に含まれる。すなわち、細胞質雄性不稔性を維持している限り、例えば、受託番号FERM BP-11506で特定されるユーストマ属植物の突然変異体や遺伝子組換え体等もその範囲に含まれる。
In the present specification, “Eustoma plants identified by the accession number FERM BP-11506” also includes plants having cytoplasmic male sterility traits that are substantially equivalent to the plants. That is, as long as cytoplasmic male sterility is maintained, for example, a mutant or gene recombinant of the Eustoma plant identified by the accession number FERMFERBP-11506 is also included in the scope.
本明細書において、「受託番号FERM BP-11507で特定されるユーストマ属植物のカルス」には、当該カルスから再生された植物が細胞質雄性不稔性の形質を有する、当該カルスと実質的に等価なカルスもその範囲に含まれる。すなわち、再生される植物が細胞質雄性不稔性を維持している限り、例えば、受託番号FERM BP-11507で特定されるユーストマ属植物のカルスに由来する突然変異体や遺伝子組換え体、ならびに細胞質雄性不稔性を維持している受託番号FERM BP-11506で特定されるユーストマ属植物の突然変異体や遺伝子組換え体等由来のカルスもその範囲に含まれる。
In the present specification, “callus of Eustoma plant identified by accession number FERM BP-11507” is substantially equivalent to the callus in which the plant regenerated from the callus has a cytoplasmic male sterility character. The callus is also included in the scope. That is, as long as the regenerated plant maintains cytoplasmic male sterility, for example, a mutant or gene recombinant derived from a callus of the Eustoma genus plant identified by accession number FERM BP-11507, and cytoplasm Callus derived from mutants of Eustoma plants identified by the accession number FERM BP-11506 maintaining male sterility or genetically modified plants are also included in the scope.
本発明に係る細胞質雄性不稔ユーストマは、次のような特徴を有する。
（１）雑種第一代交配品種を作出する際に種子親の除雄が不要であるため、手間がかからず、経済的な採種ができる。
（２）雄性可稔系統と交配した場合、後代は雄性不稔性を有するため、純度の高い種子を供給することができる。
（３）当該系統と核ゲノムが同一であって細胞質が正常である維持系統との交配により、系統の維持および増殖が容易である。
（４）自家受粉しないため、受粉による花の老化を抑制することができ、その結果、花持ちがよい。
（５）花粉を有さないため、花粉の飛散によって花弁や衣服を汚損することがない。 The cytoplasmic male sterile Eustoma according to the present invention has the following characteristics.
(1) When producing a hybrid first-generation hybrid cultivar, it is not necessary to remove the seed parent, so it is not time-consuming and economical seeding is possible.
(2) When crossed with a male fertile line, the progeny have male sterility, so that seeds with high purity can be supplied.
(3) The strain can be easily maintained and propagated by mating with a maintenance strain having the same nuclear genome as that of the strain and a normal cytoplasm.
(4) Since it does not self-pollinate, it can suppress the aging of the flower by pollination and, as a result, has a long flower.
(5) Since there is no pollen, petals and clothes will not be polluted by pollen scattering.
（１）雑種第一代交配品種を作出する際に種子親の除雄が不要であるため、手間がかからず、経済的な採種ができる。
（２）雄性可稔系統と交配した場合、後代は雄性不稔性を有するため、純度の高い種子を供給することができる。
（３）当該系統と核ゲノムが同一であって細胞質が正常である維持系統との交配により、系統の維持および増殖が容易である。
（４）自家受粉しないため、受粉による花の老化を抑制することができ、その結果、花持ちがよい。
（５）花粉を有さないため、花粉の飛散によって花弁や衣服を汚損することがない。 The cytoplasmic male sterile Eustoma according to the present invention has the following characteristics.
(1) When producing a hybrid first-generation hybrid cultivar, it is not necessary to remove the seed parent, so it is not time-consuming and economical seeding is possible.
(2) When crossed with a male fertile line, the progeny have male sterility, so that seeds with high purity can be supplied.
(3) The strain can be easily maintained and propagated by mating with a maintenance strain having the same nuclear genome as that of the strain and a normal cytoplasm.
(4) Since it does not self-pollinate, it can suppress the aging of the flower by pollination and, as a result, has a long flower.
(5) Since there is no pollen, petals and clothes will not be polluted by pollen scattering.
２．細胞質雄性不稔ユーストマの作出方法
本発明に係る雄性不稔ユーストマの作出方法においては、雌親としてEustoma属植物の野生種、雄親としてEustoma grandiflorumとを交配し、これらの交雑後代の中から雄性不稔性を示す個体を選抜して育成する。雄性不稔性を示す個体としては、雄ずいが全く存在しない、又は雄ずいの発達が不完全な状態の個体を選抜する。更に、雄性不稔性が細胞質遺伝する形質であることを確認するため、雄性可稔系統であるユーストマを戻し交配し、後代植物が雄性不稔性を示すことを確認する。 2. Method for producing cytoplasmic male sterile Eustoma In the method for producing male sterile Eustoma according to the present invention, a wild species of a plant of the genus Eustoma is mated as a female parent and Eustoma grandiflorum as a male parent. Individuals showing sterility are selected and nurtured. As an individual showing male sterility, an individual having no stamen or incomplete stamen development is selected. Furthermore, in order to confirm that male sterility is a trait inherited by cytoplasm, the male sterilized strain Eustoma is backcrossed to confirm that the progeny plant exhibits male sterility.
本発明に係る雄性不稔ユーストマの作出方法においては、雌親としてEustoma属植物の野生種、雄親としてEustoma grandiflorumとを交配し、これらの交雑後代の中から雄性不稔性を示す個体を選抜して育成する。雄性不稔性を示す個体としては、雄ずいが全く存在しない、又は雄ずいの発達が不完全な状態の個体を選抜する。更に、雄性不稔性が細胞質遺伝する形質であることを確認するため、雄性可稔系統であるユーストマを戻し交配し、後代植物が雄性不稔性を示すことを確認する。 2. Method for producing cytoplasmic male sterile Eustoma In the method for producing male sterile Eustoma according to the present invention, a wild species of a plant of the genus Eustoma is mated as a female parent and Eustoma grandiflorum as a male parent. Individuals showing sterility are selected and nurtured. As an individual showing male sterility, an individual having no stamen or incomplete stamen development is selected. Furthermore, in order to confirm that male sterility is a trait inherited by cytoplasm, the male sterilized strain Eustoma is backcrossed to confirm that the progeny plant exhibits male sterility.
３．雑種第一代種子の作出方法
本発明の方法で作出した細胞質雄性不稔ユーストマに、優良な形質を有するユーストマ属植物を連続戻し交配することにより、後代として、細胞質雄性不稔性を有する優良系統を得ることができる。この細胞質雄性不稔性を有する優良系統は、雑種第一代種子（Ｆ１種子）を得るための種子親として使用することができる。 3. Production method of hybrid first-generation seeds Excellent lines having cytoplasmic male sterility as progenies by continuously backcrossing Eustoma plants having excellent traits to cytoplasmic male sterile Eustoma produced by the method of the present invention Can be obtained. This superior line having cytoplasmic male sterility can be used as a seed parent for obtaining a hybrid first-generation seed (F1 seed).
本発明の方法で作出した細胞質雄性不稔ユーストマに、優良な形質を有するユーストマ属植物を連続戻し交配することにより、後代として、細胞質雄性不稔性を有する優良系統を得ることができる。この細胞質雄性不稔性を有する優良系統は、雑種第一代種子（Ｆ１種子）を得るための種子親として使用することができる。 3. Production method of hybrid first-generation seeds Excellent lines having cytoplasmic male sterility as progenies by continuously backcrossing Eustoma plants having excellent traits to cytoplasmic male sterile Eustoma produced by the method of the present invention Can be obtained. This superior line having cytoplasmic male sterility can be used as a seed parent for obtaining a hybrid first-generation seed (F1 seed).
４．花持ち試験
本発明に係る細胞質雄性不稔ユーストマの花持ちの評価は、以下のようにして実施することができる。なお、本明細書中において「花持ち」とは、「開花した花の維持」を意味し、よって、「花持ちが良い」とは、「開花始めから開花終わりまでの開花期間が相対的に長いこと」を意味する。ユーストマの開花期間が相対的に長いかどうかは、通常の雄性可稔ユーストマと細胞質雄性不稔ユーストマを、同一環境条件下で複数個体を栽培して比較評価することにより判断される。 4). Flower endurance test The evaluation of the endurance of cytoplasmic male sterile Eustoma according to the present invention can be carried out as follows. In the present specification, “flower holding” means “maintenance of a flower that has blossomed”, and therefore “flower holding is good” means that the flowering period from the beginning of flowering to the end of flowering is relatively Means "long". Whether or not the flowering period of the eustoma is relatively long is determined by cultivating a plurality of individual male fertile and cytoplasmic male sterile eustomas under the same environmental conditions for comparative evaluation.
本発明に係る細胞質雄性不稔ユーストマの花持ちの評価は、以下のようにして実施することができる。なお、本明細書中において「花持ち」とは、「開花した花の維持」を意味し、よって、「花持ちが良い」とは、「開花始めから開花終わりまでの開花期間が相対的に長いこと」を意味する。ユーストマの開花期間が相対的に長いかどうかは、通常の雄性可稔ユーストマと細胞質雄性不稔ユーストマを、同一環境条件下で複数個体を栽培して比較評価することにより判断される。 4). Flower endurance test The evaluation of the endurance of cytoplasmic male sterile Eustoma according to the present invention can be carried out as follows. In the present specification, “flower holding” means “maintenance of a flower that has blossomed”, and therefore “flower holding is good” means that the flowering period from the beginning of flowering to the end of flowering is relatively Means "long". Whether or not the flowering period of the eustoma is relatively long is determined by cultivating a plurality of individual male fertile and cytoplasmic male sterile eustomas under the same environmental conditions for comparative evaluation.
花持ち試験の一例として、切花後の花持ちを評価する方法について説明する。この方法では、まず試験材料として、同一条件で栽培したユーストマのうち、通常の雄性可稔個体と雄性不稔個体をそれぞれ適当数用意する。各ユーストマは開花日（花弁の開きが認められた日）当日ほぼ同時に、花柄をつけた状態で採花し、花柄部分を一定の長さ（例えば、4～6cm程度）に調整する。次いで、調整した試験材料は、茎の断面が水に浸たる状態にして、一定温度（好ましくは、18～22℃）、一定湿度（好ましくは、55～65％）、かつ明暗が12時間周期で切り替わる恒温室内に静置し、花の外観変化を観察する。
As an example of the flower endurance test, a method for evaluating the endurance after cut flowers will be described. In this method, first, an appropriate number of normal male fertile individuals and male sterile individuals are prepared as test materials, among the easterns cultivated under the same conditions. Each individual is flowered with a floral pattern at the same time on the flowering day (the day when the petal opening is recognized), and the floral part is adjusted to a certain length (for example, about 4 to 6 cm). The prepared test material is then immersed in water with the stem cross-section immersed in water, at a constant temperature (preferably 18-22 ° C.), constant humidity (preferably 55-65%), and brightness is 12 hours. Leave in a temperature-controlled room where you can switch between and observe the appearance change of flowers.
「開花始め」を花弁が開いたときと定義し、また「開花終わり」は花弁の外観（花弁のしわや萎れ）から判断・決定し、花の開花期間（花の開花始めから開花終わりまでの期間）を測定する。通常の雄性可稔個体と雄性不稔個体のそれぞれについて、測定された開花期間の平均値を求めて比較すれば、本発明の雄性不稔ユーストマの花持ちを評価することができる。
“Beginning of flowering” is defined as the time when the petal has opened, and “End of flowering” is determined and determined from the appearance of the petals (wrinkles and wrinkles of the petals), and the flowering period (from the beginning of flowering to the end of flowering) Period). If the average value of the measured flowering period is calculated | required and compared about each of a normal male fertile individual and a male sterile individual, the lasting of the male sterile Eustoma of this invention can be evaluated.
５．分子マーカーによる標識因子の作製
本発明により作製した細胞質雄性不稔ユーストマ系統と、従来知られたユーストマ属植物の間でミトコンドリアゲノムの配列を比較し、細胞質雄性不稔ユーストマ系統に特異的な領域を同定することで、標識因子となる分子マーカーを作製することができる。分子マーカーの検出は、ＰＣＲ法等、当業者に周知の方法に従って行うことができる。分子マーカーを使用することにより、本発明による細胞質雄性不稔ユーストマ系統が、形態的特徴のみならず、分子生物学的特徴によっても、従来知られたユーストマ属植物とは明確に異なる系統であることを証明することができる。 5. Preparation of labeling factor by molecular marker The sequence of mitochondrial genome is compared between cytoplasmic male sterile Eustoma strains prepared according to the present invention and the conventionally known Eustoma plants, and regions specific to cytoplasmic male sterile Eustoma strains are identified. By identifying, a molecular marker that becomes a labeling factor can be prepared. The detection of the molecular marker can be performed according to a method well known to those skilled in the art, such as a PCR method. By using molecular markers, the cytoplasmic male sterile Eustoma line according to the present invention is clearly different from conventionally known Eustoma plants not only by morphological characteristics but also by molecular biological characteristics. Can prove.
本発明により作製した細胞質雄性不稔ユーストマ系統と、従来知られたユーストマ属植物の間でミトコンドリアゲノムの配列を比較し、細胞質雄性不稔ユーストマ系統に特異的な領域を同定することで、標識因子となる分子マーカーを作製することができる。分子マーカーの検出は、ＰＣＲ法等、当業者に周知の方法に従って行うことができる。分子マーカーを使用することにより、本発明による細胞質雄性不稔ユーストマ系統が、形態的特徴のみならず、分子生物学的特徴によっても、従来知られたユーストマ属植物とは明確に異なる系統であることを証明することができる。 5. Preparation of labeling factor by molecular marker The sequence of mitochondrial genome is compared between cytoplasmic male sterile Eustoma strains prepared according to the present invention and the conventionally known Eustoma plants, and regions specific to cytoplasmic male sterile Eustoma strains are identified. By identifying, a molecular marker that becomes a labeling factor can be prepared. The detection of the molecular marker can be performed according to a method well known to those skilled in the art, such as a PCR method. By using molecular markers, the cytoplasmic male sterile Eustoma line according to the present invention is clearly different from conventionally known Eustoma plants not only by morphological characteristics but also by molecular biological characteristics. Can prove.
本発明の細胞質雄性不稔ユーストマ系統を識別するための分子マーカーとして、配列番号１および配列番号２に示した塩基配列を使用することができる。また、配列番号１または配列番号２に記載された塩基配列において1若しくは数個の塩基が欠失、置換、若しくは付加された塩基配列を有するユーストマ系統も、当該塩基配列が配列番号１または配列番号２の塩基配列と同様に増幅され、検出される限り、本発明の細胞質雄性不稔ユーストマ系統と実質的に同一な系統とみなすことができる。例えば、細胞質雄性不稔ユーストマ系統を識別するための分子マーカーとして、配列番号１または配列番号２に記載した塩基配列と80％、好ましくは90％、より好ましくは95％以上の相同性を有する塩基配列を使用することができる。
The nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 can be used as molecular markers for identifying the cytoplasmic male sterile Eustoma strain of the present invention. In addition, Eustoma strains having a base sequence in which one or several bases are deleted, substituted, or added in the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 are also included in SEQ ID NO: 1 or SEQ ID NO: As long as it is amplified and detected in the same manner as the base sequence of No. 2, it can be regarded as a strain substantially identical to the cytoplasmic male sterile Eustoma strain of the present invention. For example, as a molecular marker for discriminating cytoplasmic male sterile Eustoma strains, bases having a homology of 80%, preferably 90%, more preferably 95% or more with the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 An array can be used.
６．細胞質雄性不稔ユーストマ系統のカルス誘導、増殖、及び再分化
本発明により作製した細胞質雄性不稔ユーストマ系統は、組織培養法により無性繁殖させることが可能である。例えば、非特許文献4に開示されている、カルスを誘導し、再分化させる方法により、無性繁殖させることができる。 6). Callus induction, growth and redifferentiation of cytoplasmic male sterile Eustoma lines Cytoplasmic male sterile Eustoma lines produced according to the present invention can be propagated asexually by tissue culture methods. For example, it can be asexually propagated by the method of inducing and redifferentiating callus disclosed inNon-Patent Document 4.
本発明により作製した細胞質雄性不稔ユーストマ系統は、組織培養法により無性繁殖させることが可能である。例えば、非特許文献4に開示されている、カルスを誘導し、再分化させる方法により、無性繁殖させることができる。 6). Callus induction, growth and redifferentiation of cytoplasmic male sterile Eustoma lines Cytoplasmic male sterile Eustoma lines produced according to the present invention can be propagated asexually by tissue culture methods. For example, it can be asexually propagated by the method of inducing and redifferentiating callus disclosed in
具体的には、温室で育成した細胞質雄性不稔ユーストマ系統の葉片を表面殺菌し、カルス誘導培地に置床して培養することにより、カルス誘導を行う。その後、形成されたカルスを再分化培地に移して培養することにより、シュートを誘導する。次いで、形成されたシュートを発根培地に移して、発根を誘導し植物体を再生させる。カルス誘導、増殖、および再分化のための培養条件は、当業者であれば、公知技術に従って適宜設定することができる。
Specifically, callus induction is performed by sterilizing the surface of leaf pieces of cytoplasmic male sterile Eustoma strains grown in a greenhouse and placing them on a callus induction medium for culture. Thereafter, the formed callus is transferred to a regeneration medium and cultured to induce shoots. Next, the formed shoot is transferred to a rooting medium to induce rooting and regenerate the plant body. Culture conditions for callus induction, growth, and redifferentiation can be appropriately set according to known techniques by those skilled in the art.
本明細書において明示的に引用される全ての特許および参考文献の内容は全て参照として本明細書に組み込まれる。また、本出願が有する優先権主張の基礎となる出願である日本特許出願2012-213296号（2012年9月27日出願）の明細書および図面に記載の内容は全て参照として本明細書に組み込まれる。
The contents of all patents and references explicitly cited herein are hereby incorporated by reference. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2012-213296 (filed on September 27, 2012), which is the application on which the priority of the present application is based, are incorporated herein by reference. It is.
下記の実施例により本発明を具体的に説明するが、本発明はこれらの実施例により何ら限定されるものではない。
The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
実施例１ 新規な雄性不稔性を有するユーストマ個体の作出
本発明の雄性不稔性を有するユーストマは、アメリカ合衆国から導入した種名が特定されていない野生のEustoma属植物と、Eustoma grandiflorumとの雑種から選抜され、株式会社サカタのタネ三郷試験場において作出された。 Example 1 Creation of a new male sterility having male sterility The male sterility of the present invention is a hybrid of a wild Eustoma genus plant whose species name was introduced from the United States and Eustoma grandiflorum. And was created at the Seed Misato Proving Ground in Sakata.
本発明の雄性不稔性を有するユーストマは、アメリカ合衆国から導入した種名が特定されていない野生のEustoma属植物と、Eustoma grandiflorumとの雑種から選抜され、株式会社サカタのタネ三郷試験場において作出された。 Example 1 Creation of a new male sterility having male sterility The male sterility of the present invention is a hybrid of a wild Eustoma genus plant whose species name was introduced from the United States and Eustoma grandiflorum. And was created at the Seed Misato Proving Ground in Sakata.
本発明におけるユーストマ個体の育成過程
これまで、株式会社サカタのタネ保有の種名が特定されていない野生のEustoma属植物10数系統と、同じく株式会社サカタのタネ保有の由来の異なる親系統（E.grandiflorum）との間で合計60組程度の雑種を作製しF1種子を得た。それらF1種子を播種し、表現型を確認した上でF1集団より適当な2、3個体を選抜し、それらを集団交配した後代（以下、「F2世代」という）種子を得た。 The process of breeding the eustoma individual in the present invention Up to now, the wild Eustoma genus plant line of 10 species of Sakata Seed possession that has not been identified, and the different parent lines derived from the Sakata Seed possession (E .grandiflorum) and about 60 pairs of hybrids were produced to obtain F1 seeds. After seeding these F1 seeds and confirming the phenotype, appropriate two or three individuals were selected from the F1 population, and their progeny (hereinafter referred to as “F2 generation”) seeds were obtained.
これまで、株式会社サカタのタネ保有の種名が特定されていない野生のEustoma属植物10数系統と、同じく株式会社サカタのタネ保有の由来の異なる親系統（E.grandiflorum）との間で合計60組程度の雑種を作製しF1種子を得た。それらF1種子を播種し、表現型を確認した上でF1集団より適当な2、3個体を選抜し、それらを集団交配した後代（以下、「F2世代」という）種子を得た。 The process of breeding the eustoma individual in the present invention Up to now, the wild Eustoma genus plant line of 10 species of Sakata Seed possession that has not been identified, and the different parent lines derived from the Sakata Seed possession (E .grandiflorum) and about 60 pairs of hybrids were produced to obtain F1 seeds. After seeding these F1 seeds and confirming the phenotype, appropriate two or three individuals were selected from the F1 population, and their progeny (hereinafter referred to as “F2 generation”) seeds were obtained.
そのF2世代種子を播種し50から100個体程度栽培し、F2集団に含まれる表現形質を確認した。その結果、ほとんどのF2集団から花径の大小、花色や花型、早晩性の分離が見られたが、その中で、株式会社サカタのタネ保有の種名が特定されていない野生のEustoma属植物の内E-1を雌親、親系統の内G-1（E.grandiflorum）を雄親として交配したF1種子(以下組Aと記す)由来のF2集団の中から、予期せぬことにこれまでEustoma属植物では知られていなかった雄性不稔形質を示す個体が複数出現した。その中から、1個体を選抜し、株式会社サカタのタネ保有の親系統4系統G-2、G-3、G-4、G-5を花粉親として交配した後代種子（F2BC1世代、それぞれ組B,C,D,Eと記す）を得た。なお、世代の表記については、混乱を避けるために、E.grandiflorumをCMS系統に戻し交配した回数を基準としてBC1、BC2、・・・と記載した。
The F2 generation seeds were sown and cultivated by about 50 to 100 individuals, and the phenotypes contained in the F2 population were confirmed. As a result, the size of flower diameter, flower color, flower type, and early and late segregation were observed from most F2 populations. Among them, the species of wild Eustoma that Sakata Seed possession was not specified. Unexpectedly, among the F2 populations derived from F1 seeds (hereinafter referred to as group A) that were crossed with E-1 of the plant as the female parent and G-1 (E. grandiflorum) of the parental line as the male parent A number of individuals exhibiting male sterility traits that were not known in the Eustoma genus have appeared. Progeny seeds (F2BC1 generation, each pair) selected from one individual and crossed with 4 parent lines G-2, G-3, G-4, G-5 owned by Sakata Seed Co., Ltd. as pollen parents B, C, D, E). In order to avoid confusion, the generations are written as BC1, BC2,... Based on the number of times E. grandiflorum was backcrossed to the CMS line.
それらの後代種子(F2BC1世代)を播種し、各系統12個体の形質を確認したところ、すべての系統および個体において、雄ずいが全く存在しない、又は雄ずいの発達が不完全な状態の雄性不稔形質を示した。組C由来のF2BC1世代の4個体(以下ms-1と記す)、組D由来のF2BC1世代の2個体(以下ms-2と記す)、組E由来のF2BC1世代の3個体(以下ms-3と記す)および4個体(以下ms-4と記す)を選抜し、ms-1に親系統G-3および親系統G-6、ms-2に親系統G-4および親系統G-7、ms-3に親系統G-5および親系統G-8、ms-4に親系統G-5および親系統G-9を花粉親として交配し、それぞれ後代種子(F2BC2世代)を得た。
After seeding those progeny seeds (F2BC1 generation) and confirming the traits of 12 individuals in each line, all lines and individuals had no male stamens, or male sterility with incomplete stamen development. Showed sputum traits. F2BC1 generation 4 individuals from set C (hereinafter referred to as ms-1), F2BC1 generation 2 individuals from set D (hereinafter referred to as ms-2), F2BC1 generation 3 individuals from set E (hereinafter ms-3) Selected) and 4 individuals (hereinafter referred to as ms-4), parent line G-3 and parent line G-6 to ms-1, parent line G-4 and parent line G-7 to ms-2, The parental line G-5 and the parental line G-8 were crossed with ms-3, and the parental line G-5 and the parental line G-9 were crossed with ms-4 as pollen parents to obtain progeny seeds (F2BC2 generation), respectively.
後代種子(F2BC2世代)を播種し、後代の表現形質を確認したところ、ms-1にG-3およびG-6を交配した系統の後代集団の一部に雄ずいの発達が不完全な状態の雄性不稔個体が見られたが、それ以外の組み合わせは、雄ずいがまったくない雄性不稔形質を示した。それらの雄性不稔個体に、約100系統の親系統を交配し、後代種子(F2BC3世代)を得た。
Progeny seeds (F2BC2 generation) were sown and the phenotypic characteristics of the progeny were confirmed. As a result, the development of the stamen was incomplete in a part of the progeny population of the line in which G-3 and G-6 were crossed to ms-1. Of male sterile individuals, but the other combinations showed male sterile characteristics with no stamens. About 100 parent lines were crossed with these male sterile individuals to obtain progeny seeds (F2BC3 generation).
後代種子(F2BC3世代)を播種し、後代の表現形質を調査したところ、雄性不稔形質を確認することができた。このことから、安定的な母系遺伝を示す細胞質雄性不稔系統の作出に成功したことが実証されたため、この細胞質をSSE-CMS細胞質と命名した。なお、安定した細胞質雄性不稔を示す上記F2BC1種子は、2012年7月20日付けで独立行政法人製品評価技術基盤機構特許生物寄託センター（茨城県つくば市東１丁目１番地１中央第６）に国際寄託されている（寄託者が付した識別のための表示08P-81S、受託番号FERM BP-11506）。
When seeds of progeny (F2BC3 generation) were sown and the phenotypes of the progeny were investigated, male sterile characteristics could be confirmed. This proved that a cytoplasmic male sterile line showing stable maternal inheritance was successfully created, and this cytoplasm was named SSE-CMS cytoplasm. The above-mentioned F2BC1 seeds showing stable cytoplasmic male sterility were founded on July 20, 2012 at the Patent Organism Depositary, National Institute of Technology and Evaluation (1st, 1st, 1st East, 1st Street, Tsukuba, Ibaraki). Deposited internationally (denotation 08P-81S for identification given by the depositor, deposit number FERM BP-11506).
実施例２ 雄性不稔性の再現性の確認
雄性不稔形質の安定的な出現を確認するため、前述のEustoma属植物E-1を雌親、親系統G-10（E.grandiflorum）を雄親として交配し、F1種子(以下組Fと記載)を採種した。前述の組Aおよび組Fを播種し、それぞれ6個体を選抜し、集団交配を行ったところ、組A由来個体は56莢で0.22g、組F由来個体は59莢で0.47gの収量でF2世代種子を得た。それぞれのF2世代種子を播種し、雄性不稔個体の出現率を調査したところ、組A由来のF2世代は92個体中19個体、組F由来のF2世代は88個体中14個体において雄性不稔形質を示した。
以上の結果により、雄性不稔形質は、安定的に出現することが示された。 Example 2 Confirmation of reproducibility of male sterility In order to confirm the stable appearance of male sterility traits, the aforementioned Eustoma genus plant E-1 was used as the female parent and the parent strain G-10 (E. grandiflorum) as the male. Mating as a parent, F1 seeds (hereinafter referred to as “set F”) were collected. The above-mentioned set A and set F were sown, and 6 individuals were selected and group mating was carried out. As a result, the set A-derived individuals were 56 kg, 0.22 g, and the set F-derived individuals were 59 kg, with a yield of 0.47 g. Generational seeds were obtained. After seeding each F2 generation seed and examining the appearance rate of male sterile individuals, F2 generation derived from set A was 19 out of 92 individuals, and F2 generation derived from set F was male sterile in 14 out of 88 individuals The traits were shown.
From the above results, it was shown that male sterility traits appear stably.
雄性不稔形質の安定的な出現を確認するため、前述のEustoma属植物E-1を雌親、親系統G-10（E.grandiflorum）を雄親として交配し、F1種子(以下組Fと記載)を採種した。前述の組Aおよび組Fを播種し、それぞれ6個体を選抜し、集団交配を行ったところ、組A由来個体は56莢で0.22g、組F由来個体は59莢で0.47gの収量でF2世代種子を得た。それぞれのF2世代種子を播種し、雄性不稔個体の出現率を調査したところ、組A由来のF2世代は92個体中19個体、組F由来のF2世代は88個体中14個体において雄性不稔形質を示した。
以上の結果により、雄性不稔形質は、安定的に出現することが示された。 Example 2 Confirmation of reproducibility of male sterility In order to confirm the stable appearance of male sterility traits, the aforementioned Eustoma genus plant E-1 was used as the female parent and the parent strain G-10 (E. grandiflorum) as the male. Mating as a parent, F1 seeds (hereinafter referred to as “set F”) were collected. The above-mentioned set A and set F were sown, and 6 individuals were selected and group mating was carried out. As a result, the set A-derived individuals were 56 kg, 0.22 g, and the set F-derived individuals were 59 kg, with a yield of 0.47 g. Generational seeds were obtained. After seeding each F2 generation seed and examining the appearance rate of male sterile individuals, F2 generation derived from set A was 19 out of 92 individuals, and F2 generation derived from set F was male sterile in 14 out of 88 individuals The traits were shown.
From the above results, it was shown that male sterility traits appear stably.
実施例３ 雄性不稔性を有するユーストマ品種の花持ち試験
雄性不稔系統と可稔系統の花持ち試験を実施した。
（1）試験材料
試験材料として用いたユーストマは、通常の雄性可稔ユーストマのF1個体と細胞質雄性不稔ユーストマのF1個体の2種類を使用した。上記2種類のユーストマは、ユーストマ栽培圃場より当日開花したと思われるユーストマの花を、花柄をつけた状態で採花し、花柄部分を4cmの長さに調整した。雄性可稔ユーストマは、輸送を想定し、柱頭に花粉を強制授粉させた。また、細胞質雄性不稔ユーストマは、雄性可稔系統の花粉を強制授粉させた個体と、授粉させなかった個体をそれぞれ試験に供試した。 Example 3 Elongation test of Eustoma varieties having male sterility Fermentation test of male sterility line and varieties were carried out.
(1) Test materials The two types of Eustoma used as test materials were F1 individuals of normal male malleable and F1 individuals of cytoplasmic male sterile Eustoma. The above two types of eustoma were collected from the eustoma field that appeared to have blossomed on the day, and the floral portion was adjusted to a length of 4 cm. The male cute Eustoma was forced to pollinate the stigma for transportation. In addition, cytoplasmic male sterile Eustoma was subjected to the test for individuals that were forcibly pollinated with male pollen pollen and for individuals that were not pollinated.
雄性不稔系統と可稔系統の花持ち試験を実施した。
（1）試験材料
試験材料として用いたユーストマは、通常の雄性可稔ユーストマのF1個体と細胞質雄性不稔ユーストマのF1個体の2種類を使用した。上記2種類のユーストマは、ユーストマ栽培圃場より当日開花したと思われるユーストマの花を、花柄をつけた状態で採花し、花柄部分を4cmの長さに調整した。雄性可稔ユーストマは、輸送を想定し、柱頭に花粉を強制授粉させた。また、細胞質雄性不稔ユーストマは、雄性可稔系統の花粉を強制授粉させた個体と、授粉させなかった個体をそれぞれ試験に供試した。 Example 3 Elongation test of Eustoma varieties having male sterility Fermentation test of male sterility line and varieties were carried out.
(1) Test materials The two types of Eustoma used as test materials were F1 individuals of normal male malleable and F1 individuals of cytoplasmic male sterile Eustoma. The above two types of eustoma were collected from the eustoma field that appeared to have blossomed on the day, and the floral portion was adjusted to a length of 4 cm. The male cute Eustoma was forced to pollinate the stigma for transportation. In addition, cytoplasmic male sterile Eustoma was subjected to the test for individuals that were forcibly pollinated with male pollen pollen and for individuals that were not pollinated.
（2）試験方法
花持ち試験は、水道水を満たした試験管に試験材料を挿し、温度20℃、湿度60％（±5％）で、一般の蛍光灯（アイライン白色ラピッド40W）のオン・オフが12時間周期で切り替わる恒温室内で行なった。なお、試験中、特に水の汚れなどは認められなかったので、水の交換は行なわなかった。 (2) Test method In the flower holding test, test materials are inserted into a test tube filled with tap water, and a general fluorescent lamp (eyeline white rapid 40W) is turned on at a temperature of 20 ° C and a humidity of 60% (± 5%).・ The test was conducted in a temperature-controlled room that switched off every 12 hours. During the test, water was not contaminated, so water was not exchanged.
花持ち試験は、水道水を満たした試験管に試験材料を挿し、温度20℃、湿度60％（±5％）で、一般の蛍光灯（アイライン白色ラピッド40W）のオン・オフが12時間周期で切り替わる恒温室内で行なった。なお、試験中、特に水の汚れなどは認められなかったので、水の交換は行なわなかった。 (2) Test method In the flower holding test, test materials are inserted into a test tube filled with tap water, and a general fluorescent lamp (eyeline white rapid 40W) is turned on at a temperature of 20 ° C and a humidity of 60% (± 5%).・ The test was conducted in a temperature-controlled room that switched off every 12 hours. During the test, water was not contaminated, so water was not exchanged.
（3）評価・判定
「開花始め」は、花弁が開いたときと定義し、また「開花終わり」は花弁の外観から育種している技術者が花のしわや萎れを見て判断し、決定した。 (3) Evaluation / judgment “Beginning of flowering” is defined as the time when petals are opened, and “End of flowering” is determined by an engineer who is breeding from the appearance of petals, judging wrinkles and wilting of flowers. did.
「開花始め」は、花弁が開いたときと定義し、また「開花終わり」は花弁の外観から育種している技術者が花のしわや萎れを見て判断し、決定した。 (3) Evaluation / judgment “Beginning of flowering” is defined as the time when petals are opened, and “End of flowering” is determined by an engineer who is breeding from the appearance of petals, judging wrinkles and wilting of flowers. did.
（4）試験結果
細胞質雄性不稔のユーストマに可稔系統の花粉を強制授粉させた場合は、雄性可稔のユーストマに比較して、花持ち性にはあまり差がなかった。そのため、使用した系統の遺伝的バックグラウンドの花持ち性への影響は認められない条件下であることが証明された。また、細胞質雄性不稔のユーストマは、花粉が産出されないため、花持ち性は雄性可稔系統と比較して平均で7日も優れることが判明した(表1)。 (4) Test results When the pollen was forcedly pollinated by a cytoplasmic male sterile Eustoma, there was not much difference in flower durability compared to the male sterile Eustoma. Therefore, it was proved that the influence of the used line on the flower durability of the genetic background was not observed. In addition, the cytoplasmic male sterile Eustoma did not produce pollen, and therefore, it was found that the flower durability was excellent on average for 7 days compared to male fertile lines (Table 1).
細胞質雄性不稔のユーストマに可稔系統の花粉を強制授粉させた場合は、雄性可稔のユーストマに比較して、花持ち性にはあまり差がなかった。そのため、使用した系統の遺伝的バックグラウンドの花持ち性への影響は認められない条件下であることが証明された。また、細胞質雄性不稔のユーストマは、花粉が産出されないため、花持ち性は雄性可稔系統と比較して平均で7日も優れることが判明した(表1)。 (4) Test results When the pollen was forcedly pollinated by a cytoplasmic male sterile Eustoma, there was not much difference in flower durability compared to the male sterile Eustoma. Therefore, it was proved that the influence of the used line on the flower durability of the genetic background was not observed. In addition, the cytoplasmic male sterile Eustoma did not produce pollen, and therefore, it was found that the flower durability was excellent on average for 7 days compared to male fertile lines (Table 1).
実施例４ SSE-CMSユーストマ系統を識別するためのマーカー作製
本発明により作製したSSE-CMSユーストマ系統は、従来の栽培種や野生種には存在しなかった表現型特性を示した。一方で、分子生物学的な側面からも、従来知られたユーストマ属植物とは異なる系統であることを証明するため、SSE-CMSユーストマ系統を識別するための分子マーカーを作製することとした。 Example 4 Preparation of Markers for Discriminating SSE-CMS Eustoma Lines SSE-CMS Eustoma lines prepared according to the present invention exhibited phenotypic characteristics that did not exist in conventional cultivated and wild species. On the other hand, from the viewpoint of molecular biology, a molecular marker for identifying the SSE-CMS Eustoma line was prepared in order to prove that it is a line different from the conventionally known Eustoma genus plant.
本発明により作製したSSE-CMSユーストマ系統は、従来の栽培種や野生種には存在しなかった表現型特性を示した。一方で、分子生物学的な側面からも、従来知られたユーストマ属植物とは異なる系統であることを証明するため、SSE-CMSユーストマ系統を識別するための分子マーカーを作製することとした。 Example 4 Preparation of Markers for Discriminating SSE-CMS Eustoma Lines SSE-CMS Eustoma lines prepared according to the present invention exhibited phenotypic characteristics that did not exist in conventional cultivated and wild species. On the other hand, from the viewpoint of molecular biology, a molecular marker for identifying the SSE-CMS Eustoma line was prepared in order to prove that it is a line different from the conventionally known Eustoma genus plant.
非特許文献5に記載されたミトコンドリアのコンセンサスプライマーを用い、SSE-CMSユーストマ系統と既存のユーストマ系統を鋳型としてPCR分析を実施したところ、増幅したDNA断片のサイズが異なるマーカーを見出すことができた。このように多型を示したマーカーの中から、nad4L/orf25及びnad7/4-5の2種類を選び、増幅断片の塩基配列を解析したところ、SSE-CMSユーストマ系統に特異的な領域を同定することができた。これらの領域を標識因子とするため、表2に示すようなプライマーを設計し、PCR分析（熱変性94℃1分、アニーリング65℃1分、伸長反応72℃1分を30サイクル繰り返した）を実施したところ、SSE-CMSユーストマ系統のみからDNA断片を増幅させる2種類の分子マーカー”Marker1”及び”Marker2”を作製することに成功した。Marker1は、プライマーorf25-F及びorf25-Rを用い、配列番号1に記載した323bp のDNA断片を増幅させるマーカーである（図３）。Marker2はプライマーnad7-F及びnad7-Rを用い、配列番号2に記載した492bpの DNA断片を増幅させるマーカーである（図３）。
Using the mitochondrial consensus primer described in Non-Patent Document 5, PCR analysis was performed using the SSE-CMS Eustoma line and the existing Eustoma line as templates, and we were able to find markers with different sizes of amplified DNA fragments. . From these polymorphic markers, nad4L / orf25 and nad7 / 4-5 were selected and analyzed for the nucleotide sequence of the amplified fragment. As a result, a region specific to the SSE-CMS Eustoma strain was identified. We were able to. In order to use these regions as labeling factors, primers as shown in Table 2 were designed, and PCR analysis (thermal denaturation 94 ° C 1 minute, annealing 65 ° C 1 minute, extension reaction 72 ° C 1 minute was repeated 30 cycles) As a result, two types of molecular markers “Marker1” and “Marker2” for amplifying DNA fragments from only the SSE-CMS Eustoma strain were successfully produced. Marker 1 is a marker that amplifies the DNA fragment of 323 bp described in SEQ ID NO: 1 using primers orf25-F and orf25-R (FIG. 3). Marker 2 is a marker that amplifies the 492 bp DNA fragment described in SEQ ID NO: 2 using primers nad7-F and nad7-R (FIG. 3).
上記手法により作製したマーカーを用い、株式会社サカタのタネが育成した既存のユーストマ系統（E.grandiflorum）190系統、及び、株式会社サカタのタネが保有しているユーストマ属野生種34系統を鋳型としてPCR試験を実施したところ、これら2種類の塩基配列を保有している系統は、SSE-CMSユーストマ系統以外に存在しないことを確認した。その試験結果の一部を図2に示す。図2（A)～(C)は、以下の条件で実験を行った。
(A) Marker1：SSE-CMSユーストマ系統に特異的なマーカー
orf25-F及びorf25-Rのプライマーを用いてPCR（熱変性94℃1分、アニーリング65℃1分、伸長反応72℃1分を30サイクル）を行い、配列番号1で示した塩基配列（323bp）を増幅した。
(B) Marker2：SSE-CMSユーストマ系統に特異的なマーカー
nad7-F及びnad7-Rのプライマーを用いてPCR（熱変性94℃1分、アニーリング65℃1分、伸長反応72℃1分を30サイクル）を行い、配列番号2で示した塩基配列（492bp）を増幅した。
(C) nad5/4-5：全てのユーストマ属植物に共通のマーカー
nad5/4及びnad5/5のプライマーを用いてPCR（熱変性94℃1分、アニーリング60℃1分、伸長反応72℃1分を30サイクル）を行い、全ての系統から約1.5ｋｂのDNA断片を増幅した。
すなわち、本発明により作製した SSE-CMSユーストマ系統が、従来知られたユーストマ属植物とは異なる系統であることが明らかとなった。 Using the marker produced by the above method, 190 existing Eustoma lines (E.grandiflorum) grown by Sakata Seed Co., Ltd. and 34 Eustoma wild species 34 lines owned by Sakata Seed Co., Ltd. as templates When a PCR test was performed, it was confirmed that there were no strains possessing these two types of base sequences other than the SSE-CMS Eustoma strain. A part of the test results is shown in FIG. 2A to 2C, the experiment was performed under the following conditions.
(A) Marker1: Marker specific to SSE-CMS Eustoma line
PCR was performed using primers of orf25-F and orf25-R (heat denaturation 94 ° C. for 1 minute, annealing 65 ° C. for 1 minute, extension reaction 72 ° C. for 1 minute for 30 cycles), and the nucleotide sequence shown in SEQ ID NO: 1 (323 bp ) Was amplified.
(B) Marker2: Marker specific to SSE-CMS Eustoma line
PCR was performed using the nad7-F and nad7-R primers (heat denaturation at 94 ° C for 1 minute, annealing at 65 ° C for 1 minute, extension reaction at 72 ° C for 1 minute for 30 cycles), and the nucleotide sequence shown in SEQ ID NO: 2 (492 bp ) Was amplified.
(C) nad5 / 4-5: a marker common to all Eustoma species
Perform PCR (thermal denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, extension reaction at 72 ° C for 1 minute for 30 cycles) using nad5 / 4 and nad5 / 5 primers, and a DNA fragment of about 1.5 kb from all strains Was amplified.
That is, it has been clarified that the SSE-CMS Eustoma line produced by the present invention is a line different from conventionally known Eustoma plants.
(A) Marker1：SSE-CMSユーストマ系統に特異的なマーカー
orf25-F及びorf25-Rのプライマーを用いてPCR（熱変性94℃1分、アニーリング65℃1分、伸長反応72℃1分を30サイクル）を行い、配列番号1で示した塩基配列（323bp）を増幅した。
(B) Marker2：SSE-CMSユーストマ系統に特異的なマーカー
nad7-F及びnad7-Rのプライマーを用いてPCR（熱変性94℃1分、アニーリング65℃1分、伸長反応72℃1分を30サイクル）を行い、配列番号2で示した塩基配列（492bp）を増幅した。
(C) nad5/4-5：全てのユーストマ属植物に共通のマーカー
nad5/4及びnad5/5のプライマーを用いてPCR（熱変性94℃1分、アニーリング60℃1分、伸長反応72℃1分を30サイクル）を行い、全ての系統から約1.5ｋｂのDNA断片を増幅した。
すなわち、本発明により作製した SSE-CMSユーストマ系統が、従来知られたユーストマ属植物とは異なる系統であることが明らかとなった。 Using the marker produced by the above method, 190 existing Eustoma lines (E.grandiflorum) grown by Sakata Seed Co., Ltd. and 34 Eustoma wild species 34 lines owned by Sakata Seed Co., Ltd. as templates When a PCR test was performed, it was confirmed that there were no strains possessing these two types of base sequences other than the SSE-CMS Eustoma strain. A part of the test results is shown in FIG. 2A to 2C, the experiment was performed under the following conditions.
(A) Marker1: Marker specific to SSE-CMS Eustoma line
PCR was performed using primers of orf25-F and orf25-R (heat denaturation 94 ° C. for 1 minute, annealing 65 ° C. for 1 minute, extension reaction 72 ° C. for 1 minute for 30 cycles), and the nucleotide sequence shown in SEQ ID NO: 1 (323 bp ) Was amplified.
(B) Marker2: Marker specific to SSE-CMS Eustoma line
PCR was performed using the nad7-F and nad7-R primers (heat denaturation at 94 ° C for 1 minute, annealing at 65 ° C for 1 minute, extension reaction at 72 ° C for 1 minute for 30 cycles), and the nucleotide sequence shown in SEQ ID NO: 2 (492 bp ) Was amplified.
(C) nad5 / 4-5: a marker common to all Eustoma species
Perform PCR (thermal denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, extension reaction at 72 ° C for 1 minute for 30 cycles) using nad5 / 4 and nad5 / 5 primers, and a DNA fragment of about 1.5 kb from all strains Was amplified.
That is, it has been clarified that the SSE-CMS Eustoma line produced by the present invention is a line different from conventionally known Eustoma plants.
実施例５ SSE-CMSユーストマ系統のカルス誘導、増殖、及び再分化
本発明により作製したSSE-CMSユーストマ系統を、組織培養法により無性繁殖させるため、温室で育成したSSE-CMSユーストマ系統の葉片を採取した。葉片を1%次亜塩素酸ナトリウム溶液で10分間表面殺菌を行ない、滅菌水ですすぎを行なった。滅菌した葉片を1.5mg/l NAAを添加したMS培地へ置床しカルスを誘導した。カルスを0.5mg/l GA3、1.5mg/l BAを添加したB5培地へ移しシュートを誘導した。形成されたシュートは植物ホルモンを含まないB5培地へ移して発根させ、植物体を再生し、雄性不稔であることを確認した。 Example 5 SSE-CMS Eustoma Line Callus Induction, Proliferation, and Redifferentiation A leaf piece of an SSE-CMS Eustoma line grown in a greenhouse to allow the SSE-CMS Eustoma line produced according to the present invention to propagate asexually by tissue culture. Were collected. The leaf pieces were surface sterilized with 1% sodium hypochlorite solution for 10 minutes and rinsed with sterile water. Sterilized leaf pieces were placed on MS medium supplemented with 1.5 mg / l NAA to induce callus. Calli were transferred to B5 medium supplemented with 0.5 mg / l GA3 and 1.5 mg / l BA to induce shoots. The formed shoots were transferred to a B5 medium containing no plant hormone and rooted to regenerate the plant body and confirmed to be male sterile.
本発明により作製したSSE-CMSユーストマ系統を、組織培養法により無性繁殖させるため、温室で育成したSSE-CMSユーストマ系統の葉片を採取した。葉片を1%次亜塩素酸ナトリウム溶液で10分間表面殺菌を行ない、滅菌水ですすぎを行なった。滅菌した葉片を1.5mg/l NAAを添加したMS培地へ置床しカルスを誘導した。カルスを0.5mg/l GA3、1.5mg/l BAを添加したB5培地へ移しシュートを誘導した。形成されたシュートは植物ホルモンを含まないB5培地へ移して発根させ、植物体を再生し、雄性不稔であることを確認した。 Example 5 SSE-CMS Eustoma Line Callus Induction, Proliferation, and Redifferentiation A leaf piece of an SSE-CMS Eustoma line grown in a greenhouse to allow the SSE-CMS Eustoma line produced according to the present invention to propagate asexually by tissue culture. Were collected. The leaf pieces were surface sterilized with 1% sodium hypochlorite solution for 10 minutes and rinsed with sterile water. Sterilized leaf pieces were placed on MS medium supplemented with 1.5 mg / l NAA to induce callus. Calli were transferred to B5 medium supplemented with 0.5 mg / l GA3 and 1.5 mg / l BA to induce shoots. The formed shoots were transferred to a B5 medium containing no plant hormone and rooted to regenerate the plant body and confirmed to be male sterile.
上記方法によって作製したカルスは、2012年7月26日付けで独立行政法人製品評価技術基盤機構特許生物寄託センター（茨城県つくば市東1丁目1番地1中央第6）に国際寄託されている（寄託者が付した識別のための表示12S-134C、受託番号FERM BP-11507）。
Callus made by the above method has been deposited internationally at the Patent Biological Depositary Center of the National Institute of Technology and Evaluation (July 26, 1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture) on July 26, 2012. 12S-134C for identification given by the person, accession number FERM BP-11507).
本発明により提供される新規な細胞質雄性不稔性を有するユーストマを利用することにより、採種性、花持ち性、鑑賞性に優れたユーストマとそのF１品種を育成し、その高品質種子の採種を行うことを可能とする。
By using the novel cytoplasmic male sterility provided by the present invention, the yeast and its F1 varieties that are excellent in seeding, flower retention and appreciation are bred and the high-quality seeds are seeded. Make it possible to do.
Claims (34)
- 細胞質雄性不稔性を有するEustoma属植物、またはその後代。 Eustoma genus plant having cytoplasmic male sterility, or its progeny.
- 配列番号１もしくは配列番号２に記載した塩基配列を有することを特徴とする、請求項１に記載のEustoma属植物、またはその後代。 The plant of the genus Eustoma according to claim 1, or a progeny thereof, which has the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2.
- 前記Eustoma属植物またはその後代が、配列番号１もしくは配列番号２に記載した塩基配列を有するEustoma属植物を種子親、任意のEustoma属植物を花粉親とした交雑後代から作出された細胞質雄性不稔植物であることを特徴とする、請求項１または２に記載のEustoma属植物、またはその後代。 A cytoplasmic male sterility produced from a progeny of the hybrid, wherein the Eustoma genus plant or its progeny is an Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 as a seed parent and an arbitrary Eustoma genus plant as a pollen parent The plant of the genus Eustoma according to claim 1 or 2, or a progeny thereof, wherein the plant is a plant.
- 前記Eustoma属植物またはその後代が、配列番号１もしくは配列番号２に記載した塩基配列を有するEustoma属植物を種子親、E. grandiflorumを花粉親とした交雑後代から作出された細胞質雄性不稔植物であることを特徴とする、請求項１～３のいずれか１項に記載のEustoma属植物、またはその後代。 A cytoplasmic male sterilized plant produced from a progeny of the cross, wherein the Eustoma genus plant or its progeny is a seed parent of the Eustoma genus plant having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 and E.２grandiflorum is the pollen parent. The plant of the genus Eustoma according to any one of claims 1 to 3, or a progeny thereof.
- 請求項１～４のいずれか１項に記載のEustoma属植物またはその後代の植物体の一部。 A part of the plant belonging to the genus Eustoma or a progeny plant according to any one of claims 1 to 4.
- 請求項１～４のいずれか１項に記載のEustoma属植物またはその後代の種子。 The plant of the genus Eustoma or a seed of its progeny according to any one of claims 1 to 4.
- 請求項１～４のいずれか１項に記載のEustoma属植物またはその後代の細胞を含むカルス。 Callus comprising a plant of the genus Eustoma or a progeny cell thereof according to any one of claims 1 to 4.
- 請求項７に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物、またはその後代。 A plant derived from the genus Eustoma derived from the callus according to claim 7 and propagated asexually by tissue culture, or a progeny thereof.
- 請求項８に記載のEustoma属植物またはその後代の植物体の一部。 A part of the plant of the genus Eustoma according to claim 8 or its progeny.
- 請求項７に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物を作製する方法。 A method for producing an Eustoma genus plant derived from the callus according to claim 7 and propagated asexually by tissue culture.
- 請求項１～４および８のいずれか１項に記載のEustoma属植物もしくはその後代、請求項５および９に記載の植物体の一部、請求項６に記載の種子、または請求項７に記載のカルスに含まれる細胞質。 A plant of the genus Eustoma or a progeny thereof according to any one of claims 1 to 4 and 8, a part of a plant body according to claims 5 and 9, a seed according to claim 6, or a claim according to claim 7. Cytoplasm contained in the callus.
- 請求項１～４および８のいずれか１項に記載のEustoma属植物もしくはその後代、請求項５および９に記載の植物体の一部、請求項６に記載の種子、または請求項７に記載のカルスに含まれるミトコンドリア。 A plant of the genus Eustoma or a progeny thereof according to any one of claims 1 to 4 and 8, a part of a plant body according to claims 5 and 9, a seed according to claim 6, or a claim according to claim 7. Mitochondria contained in callus.
- 受託番号FERM BP-11506で特定される、細胞質雄性不稔性を有するEustoma属植物、またはその後代。 A plant belonging to the genus Eustoma having cytoplasmic male sterility, or its progeny, identified by accession number FERM そ の 後 BP-11506.
- 請求項１３に記載のEustoma属植物またはその後代の植物体の一部。 A part of the plant of the genus Eustoma according to claim 13 or its progeny.
- 請求項１３に記載のEustoma属植物またはその後代の種子。 The plant of the genus Eustoma or a seed of its progeny according to claim 13.
- 請求項１３に記載のEustoma属植物もしくはその後代、請求項１４に記載の植物体の一部、または請求項１５に記載の種子に含まれる細胞質。 The cytoplasm contained in the plant of the genus Eustoma or a progeny thereof according to claim 13, a part of the plant body according to claim 14, or the seed according to claim 15.
- 請求項１３に記載のEustoma属植物もしくはその後代、請求項１４に記載の植物体の一部、または請求項１５に記載の種子に含まれるミトコンドリア。 A mitochondrion contained in a plant of the genus Eustoma or a progeny thereof according to claim 13, a part of a plant body according to claim 14, or a seed according to claim 15.
- 受託番号FERM BP-11507で特定される、細胞質雄性不稔性を有するEustoma属植物のカルス。 Callus of Eustoma genus plant having cytoplasmic male sterility identified by accession number FERM BP-11507.
- 請求項１８に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物、またはその後代。 A plant derived from the genus Eustoma derived from the callus according to claim 18 and propagated asexually by tissue culture, or a progeny thereof.
- 請求項１９に記載のEustoma属植物またはその後代の植物体の一部。 A part of the plant of the genus Eustoma according to claim 19 or its progeny.
- 請求項１８に記載のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物を作製する方法。 A method for producing an Eustoma genus plant derived from the callus according to claim 18 and asexually propagated by tissue culture.
- 請求項１８に記載のカルス、請求項１９に記載のEustoma属植物もしくはその後代、または請求項２０に記載の植物体の一部に含まれる細胞質。 A cytoplasm contained in a callus according to claim 18, a plant belonging to the genus Eustoma or a progeny thereof according to claim 19, or a part of a plant body according to claim 20.
- 請求項１８に記載のカルス、請求項１９に記載のEustoma属植物もしくはその後代、または請求項２０に記載の植物体の一部に含まれるミトコンドリア。 A callus according to claim 18, a plant belonging to the genus Eustoma or a progeny thereof according to claim 19, or a mitochondria contained in a part of a plant body according to claim 20.
- 請求項１～４および８のいずれか１項に記載のEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。 A plant of the genus Eustoma or a progeny thereof according to any one of claims 1 to 4 and 8 is used as a seed parent, and a plant of the genus Eustoma that can be crossed with the plant is crossed as a pollen parent. A method for producing a hybrid first-generation seed, comprising seeding a first-generation seed.
- 請求項１３に記載のEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。 14. The Eustoma genus plant according to claim 13 or its progeny is used as a seed parent, the Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent, and the hybrid first seed is collected from the mated seed parent. , How to make hybrid seeds.
- 請求項１８に記載のカルスより再生させたEustoma属植物またはその後代を種子親とし、該植物と交配可能なEustoma属植物を花粉親として交配し、交配後の種子親から雑種第一代種子を採種することを含む、雑種第一代種子の作出方法。 An Eustoma genus plant regenerated from the callus according to claim 18 or a progeny thereof is used as a seed parent, an Eustoma genus plant that can be crossed with the plant is crossed as a pollen parent, and a hybrid first seed is obtained from the mated seed parent. A method for producing hybrid primary seeds, including seeding.
- 請求項２４～２６のいずれか１項に記載の方法により作出された雑種第一代種子。 A hybrid first-generation seed produced by the method according to any one of claims 24 to 26.
- 請求項２７に記載の雑種第一代種子から生育させた雑種第一代植物。 A hybrid primary plant grown from the hybrid primary seed according to claim 27.
- 配列番号１もしくは配列番号２に記載した塩基配列を細胞質に有するEustoma属植物と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。 Eustoma genus plant having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the cytoplasm and Eustoma genus plant having useful traits are continuously backcrossed to have the aforementioned useful traits and express cytoplasmic male sterility A method for producing a plant belonging to the genus Eustoma,
- 受託番号FERM BP-11506で特定される、細胞質雄性不稔性を有するEustoma属植物またはその後代と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。 The Eustoma genus plant having cytoplasmic male sterility or its progeny identified by the accession number FERM BP-11506 and the Eustoma genus plant having a useful trait are continuously backcrossed to have the useful trait, and the cytoplasmic male A method for producing a plant of the genus Eustoma, characterized by expressing sterility.
- 受託番号FERM BP-11507で特定される、細胞質雄性不稔性を有するEustoma属植物のカルスから誘導され、組織培養により無性繁殖されるEustoma属植物またはその後代と、有用形質を有するEustoma属植物を連続戻し交雑することにより、前記有用形質を有し、細胞質雄性不稔性を発現させることを特徴とするEustoma属植物の作出方法。 Eustoma genus plant or its progeny derived from callus of Eustoma genus plant having cytoplasmic male sterility and identified as accession number FERM BP-11507, and its progeny, and Eustoma genus plant having useful traits A method for producing a plant of the genus Eustoma having the above-mentioned useful traits and expressing cytoplasmic male sterility by continuous backcrossing.
- 有用形質を有するEustoma属植物がE. grandiflorumに由来するものである、請求項２９～３１のいずれか１項に記載の作出方法。 The production method according to any one of claims 29 to 31, wherein the plant belonging to the genus Eustoma having a useful trait is derived from E. grandiflorum.
- 請求項２９～３２のいずれか１項に記載の方法により作出されたEustoma属植物、またはその後代。 A plant belonging to the genus Eustoma produced by the method according to any one of claims 29 to 32, or a progeny thereof.
- 請求項３３に記載のEustoma属植物またはその後代の植物体の一部。 A part of the plant of the genus Eustoma according to claim 33 or its progeny.
Priority Applications (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2882797A CA2882797C (en) | 2012-09-27 | 2013-09-26 | Cytoplasmic male sterile eustoma and a method for developing thereof |
| RU2015111151A RU2694957C2 (en) | 2012-09-27 | 2013-09-26 | Plants of genus eustoma with cytoplasmic male sterility and method of their introduction |
| CN201380050323.5A CN104853595B (en) | 2012-09-27 | 2013-09-26 | Cytoplasmic male sterility Lisianthus and its manufacture method |
| JP2014538193A JP5841263B2 (en) | 2012-09-27 | 2013-09-26 | Cytoplasmic male sterile eustoma and production method thereof |
| US14/431,913 US9504215B2 (en) | 2012-09-27 | 2013-09-26 | Cytoplasmic male sterile eustoma and a method for developing thereof |
| AU2013321900A AU2013321900B2 (en) | 2012-09-27 | 2013-09-26 | Cytoplasmic male sterile eustoma and a method for developing thereof |
| AP2015008262A AP2015008262A0 (en) | 2012-09-27 | 2013-09-26 | Cytoplasmic male sterile eustoma and a method for developing thereof |
| EP13841979.1A EP2901852A4 (en) | 2012-09-27 | 2013-09-26 | EUSTOMA HAVING CYTOPLASMIC MALE STERILITY AND PROCESS FOR PRODUCING THE SAME |
| CR20150116A CR20150116A (en) | 2012-09-27 | 2015-03-06 | EUSTOMA WITH CYTOPLASM MALE STERILITY AND METHOD TO DEVELOP THE SAME |
| IL237929A IL237929A (en) | 2012-09-27 | 2015-03-25 | Cytoplasmic male sterile eustoma and a method for developing thereof |
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| JP2012-213296 | 2012-09-27 | ||
| JP2012213296 | 2012-09-27 |
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| US (1) | US9504215B2 (en) |
| EP (1) | EP2901852A4 (en) |
| JP (1) | JP5841263B2 (en) |
| CN (1) | CN104853595B (en) |
| AP (1) | AP2015008262A0 (en) |
| AU (1) | AU2013321900B2 (en) |
| CA (1) | CA2882797C (en) |
| CL (1) | CL2015000777A1 (en) |
| CR (1) | CR20150116A (en) |
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| RU (1) | RU2694957C2 (en) |
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| CN110358856B (en) * | 2019-07-23 | 2023-04-07 | 四川省农业科学院生物技术核技术研究所 | Amplification primer of pleurotus mitochondrial nad4L gene and application thereof in identification of pleurotus species |
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| US5530191A (en) * | 1994-03-24 | 1996-06-25 | Rutgers, The State University Of New Jersey | Method for producing cytoplasmic male sterility in plants and use thereof in production of hybrid seed |
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| JP2004000103A (en) * | 2001-08-03 | 2004-01-08 | Noboru Sase | Eustoma having variant pistil and method for breeding the same |
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Also Published As
| Publication number | Publication date |
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| EP2901852A4 (en) | 2016-04-13 |
| AU2013321900B2 (en) | 2017-10-26 |
| AP2015008262A0 (en) | 2015-02-28 |
| CN104853595A (en) | 2015-08-19 |
| AU2013321900A1 (en) | 2015-02-19 |
| JP5841263B2 (en) | 2016-01-13 |
| TWI659689B (en) | 2019-05-21 |
| CA2882797A1 (en) | 2014-04-03 |
| RU2694957C2 (en) | 2019-07-18 |
| US9504215B2 (en) | 2016-11-29 |
| CL2015000777A1 (en) | 2015-07-10 |
| JPWO2014050116A1 (en) | 2016-08-22 |
| EP2901852A1 (en) | 2015-08-05 |
| CR20150116A (en) | 2015-08-14 |
| CA2882797C (en) | 2017-07-11 |
| CN104853595B (en) | 2017-07-11 |
| US20150272020A1 (en) | 2015-10-01 |
| TW201427592A (en) | 2014-07-16 |
| IL237929A (en) | 2016-05-31 |
| RU2015111151A (en) | 2016-11-20 |
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